GB2513024A - Compositions and kits for molecular counting - Google Patents

Compositions and kits for molecular counting Download PDF

Info

Publication number
GB2513024A
GB2513024A GB1408829.8A GB201408829A GB2513024A GB 2513024 A GB2513024 A GB 2513024A GB 201408829 A GB201408829 A GB 201408829A GB 2513024 A GB2513024 A GB 2513024A
Authority
GB
United Kingdom
Prior art keywords
labeled
molecule
molecules
paragraph
tag
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
GB1408829.8A
Other versions
GB201408829D0 (en
GB2513024B (en
Inventor
Glenn K Fu
Stephen P A Fodor
Julie Wilhelmy
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cellular Research Inc
Original Assignee
Cellular Research Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cellular Research Inc filed Critical Cellular Research Inc
Publication of GB201408829D0 publication Critical patent/GB201408829D0/en
Publication of GB2513024A publication Critical patent/GB2513024A/en
Application granted granted Critical
Publication of GB2513024B publication Critical patent/GB2513024B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1065—Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/10—Nucleotidyl transfering
    • C12Q2521/107—RNA dependent DNA polymerase,(i.e. reverse transcriptase)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/50—Other enzymatic activities
    • C12Q2521/531—Glycosylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/155—Modifications characterised by incorporating/generating a new priming site
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/173—Modifications characterised by incorporating a polynucleotide run, e.g. polyAs, polyTs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/179—Modifications characterised by incorporating arbitrary or random nucleotide sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2539/00—Reactions characterised by analysis of gene expression or genome comparison
    • C12Q2539/10—The purpose being sequence identification by analysis of gene expression or genome comparison characterised by
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2545/00—Reactions characterised by their quantitative nature
    • C12Q2545/10—Reactions characterised by their quantitative nature the purpose being quantitative analysis
    • C12Q2545/114—Reactions characterised by their quantitative nature the purpose being quantitative analysis involving a quantitation step
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/50—Detection characterised by immobilisation to a surface
    • C12Q2565/514—Detection characterised by immobilisation to a surface characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Plant Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)

Abstract

A method for quantifying a nucleic acid in an original sample wherein the nucleic acids in the original sample are tagged with oligonucleotides to form a plurality of labelled-nucleic acid molecules, the treating the sample with a uracil DNA glycosylase (UDG), amplifying these treated labelled nucleic acid molecules and quantifying a nucleic acid molecule in the sample. The method may produce a plurality of labelled amplicons with a unique tag identifier regions. Alternatively claimed is a clonal amplification method comprising stochastically labelling a plurality of molecules with a plurality of oligonucleotide tags, wherein the plurality of molecules comprises at least two different sequences, the plurality of tags comprises at least 2 different oligonucleotide sequences, the labelled molecules are amplified to produce labelled amplicons, the labelled amplicons are amplified by nested PCR to produce a plurality of nested PCR amplified labelled amplicons and detecting said plurality of nested PCR labelled amplicons.

Description

COMPOSITO\S AND KITS FOR \IOI FCfl -R COIN FING
CROSS-REFERENC.:E TO RELATED APPiJCATONS
(00011 l'hi& appIicatic'i claims t& bcnctit oftJ$. Prc'visiomd i\pplIC.atiCli No. 61 /6O*39 I fikd Fcbruary27 2()12 and U.S. Iro'ion& AjptCttt.1Ofl NO. .&V74$J8$.tikd Decembvr 21. 201.2., bethel vhich we idlcorpSted. by rdferchee blerbin mn.t.hcir enthuy.
FiflhloIF raE INVENTION {DOO2 Method', and USC\ oltrotxt La eountin. tte thseosed s4otecukt\ Afl be couLed liv sC.qw cin" and i ra It inn h nuolbel u or eti Lrc c a. hu no k ru te Mo tithe all also be counted b hyhidization of the tholecule to a: solid. support and.deteedon of the h'hrdized moIeduks. lit sOme instatice, thd. niolecules: to Ic counted tft Iãhckid The molecules tO be counted may also be amplified.
BACKGROUND OF ThE INVENTION
[OOO3 Accurate &tetrti±natk)i &theqfiSitv of nucleic acids j necesry ic t wide variety otelinicat and roseardh nieaure.meius, When dLssoived irs&uhn the average.
concentration a.ni eLe ac'ds R'\A oi D\A) can he determined ov IJV ght ab'>oibancc spectrophotometrv or by fluomseent DNAA.indin stains. However, the measurement reqdircd isoften not just kr the total.amouito.fnucleid acids prosent but.specif9call.y br one W molt 4iCIL ul lntifl tY&t cOoulntd nd on ed th dl oft ic oUc rut etc acah t im the sirnk Ir thcc the huLcC iud iuktuie of ujtre%t I\ u\uaiI\ chtngtu'hed fun i of the oth er hnc.lebz uckk tbtough a ddfbed.suquence< of nuchuotide' that u.dique to thu species ot interest. A short synthetic dbo er d.eoxvribo-oh oruceotide with a cotrqilcnicritary seqticLOc:e o the nucleic: acid of interest can be ised fbr irs.detectkut and identifiiät.idtl. For in.$áncd, tho1yre*ra. Chthn R*etS (PCR). usus a pair of these oiigonucIeoticles to scrve s anr!etihiug p mersthr repeated eycks bFhNApoEyrned.atkm mectated e CIA pobiucaasc cnz)mcs D\'\ imcroan'n axe anothet conmon d:reurnn method where ol.io:nucicotides are. immobilized on solid supports o hybridize to. DNA mci ecules bea.mij compkrrentar' eluentes Ahhouh both PC R nd flflCTOJltt) nethoL, are capable. of spcdtIc. detection3 aecuratd d.eter.thintitid.n of the quantity of the d.ctdet&t e diliictdt spc wI s v4 en II l' present 1 Jo anmdiuxt or I en cot talflCL' sithm a Earec haekeround ofother Itudek iws II' the ese of PCR (abo soiretimes i cten ed to a' quckthta L%C -PC R c PC R. faq i& i. or t.akwnc PC R, the dmount cit niph lied DNA molecuks represents. an estimate of its concentration to the slaflmg sohthoti. En the ease of nueroarrays. thc amthuit of DNA hfrridized is an esthSte: of ft icentration in sOlutioft.
In. bOth. ca:sc& only reIati.vc TTIca.1uvei.ne.nts Ut etmeentratiun. Cfl. X.. .nade and iDjm&r)f coftis &fnu.c.lek.acid. iii the safli:Øk ta.mot be ØeeIseIt dtentithed..IitWen,.
v% .Yi n. tL flCt' Ut t'EC Cid\ UI jt -dL tj flfflfjj enncuItrdhen' a e fl'kicitti UI ft tt,t retiv: eemparisoiis C4t,.b nad tothIs st andard re.fé.re.ice. to estimate. the.thsclute nu.mhier of*c5pies OfflLC.k{C &idS bi:g ktécted.
UHW4J Dtg:ai PCR s unt. nieth&1 that can b ua to trsnni t<e absc' ute rirnhei of' D1A fflQICCUkS C.. a pariietdar nucløtuje equ.nce (Syke t. Li BiOtCchflk1u& 3;: .414-4..1) (1992:). Vogelstchi ci at. I)igitál PCR. Pree.Nfl Acad Sd hA 96S 9216-:4l (1999)), hi this method, the. nuclek ad4. solution is diluted and stoehatkaR partitioned into individ.udl centame o that Ibue Is on aw'atte k than Nv.. moket fe in sei" %\u cnnruue N K is then uscd:to detect., the prescnce of:F:e nucleic acid molecule of interesU in: It quantitative partitioning is ssumed, the d3inamic range i5:gOverneCt by the: number of ceniaiheis avai idhie.kt stochastic separation. NI ieio fid*jc.htion. and çdco tc'rsized enthiióh droplets: can be used to increase tbc number ofrorim.inas available, Uw. ebv ext.ending..the me4nutenic'i dnauc iange (hu et a Am.1 Ubet Gvnecol200 541 cSfl-5 17 2009 Kaiinima et al. Nucleic Acids Rca. 25: 1 999'2004 (1997).).. Due. to the p..hyaleal cotistrail ts of i.n.a.nufa.ctunng huge number,s of separate containers and *iv can'ying out. these larger numbers in putetice the digital PCR doethod is tin'dtdd to inveStiations on only a annul.
ol tht\ e i D\ i mulct uk at a Inn' t00051 Recently. a teW method t dctermthe the absolute quantity of DNA molecules has..
been den'xmstnded where identical copi of' idividna.) DNA rnol.eouks' can be counted alter the.stochastic: attachmeni of a set of diverse nucleic add labels (Pu et al., ProeNati Acad Sc.Ii USA 108:. &02t903l (2011)), Unlihe digitil PUt, this isa hiih1y piuEáflel. tnetho.d cajabk of cone ig many du1fecnt fl\ \ tnokcwes nnn hancu rds fri I n ho& each cop" ot a moei Uk rmtontl a. at±e\ l a short nuekic acid Libel h choosing ion a MflLC non depte.tiIn&,r.eservoii' of diverse labels. The subsequent diversity of the labeled mc:lecu!es is ttos:ctned by the stuns s of nu,don'i coo Ice ed depends on the uambe of cop.ie of idenucal moiecu.les ii. the collection compared number of kinds of labels (Thee the molecules.
arc. labeled. they can be amplified so that simple present/absent threshold. deteenon methods can be u'"d toi et h Cc.jnunua the number of tju4u. ll abded t iigcs revea the ongw ii atter of.flolec.uk.s of.each specS. tNike di intl PCR, which sochaticalle.xpai* ckntied n olctue into pbysLal epar:, the nv.thod of toehatt hbdi ig \rJaijs tdc ita 2 niocctdcs nto hc'nucil ce \r irnportao dMtmetion fun digital PC ft is that the stocoastie labelwe method does no tequre the challen4 ng pbysikat ceparenor of atenticat I.flc3 cu1cs n dis dud phvaLal twjt un,j The a1ptotch r. r:tcaL and ait iai. ing.
dffipie dawthr device such is a ciThirny with chrnp1me:ntafli pmhe euente th the aheis Sn he. used. u.iIdenxit, imd thtint the number: of lathels peaeikL i:t add.itioli wheP stochastic laheis arc:atiache.d to DNA molecules that arc prepared flIt DNA sequencing rSdouts, the hthehrig sdquenc darrsdrve as discreet: counting tans. for áh.sbiut quantitation, s una ie ide tthe.' to distinwh 1.4011 Ol3hi ill> tagged:en plat. from us arnpLfc'd daughtCt.mokcuks ik.in&a;i. si. Proc Nail Aend Sci USA 108: :530953.5: 2.0 f)).
SUMMARY OF TtW N\ F\TION
[00061 In owe cqihedimcnt s a dun'l e'etn l lnscupnon mrtholJ (omfibing U eeflLctuu4 a imple <npt nont. p1w dt) ot' RN \ mu ecu a plulal t" O oligonuclootido lags to paduce iabekdRNA ntoleclile. rehereth:. the piurality. oPRNA rue keules comprise at Idast. 2.mRNA molecules o: different sequences; the.: plurality of oh onud.eotit1eaegs compi e5.at. k.tist.2 o]igcdmdeot.ide ttgs of difftnt ieqbcndes; ind. the phlnhi) o oligom ekouth la"> Oii pi hI S 1111 0hfl1 F sap.h. ic), bC oIidhL Hug fh' ti md sytuhesis eactien by contacting the iahckd-RNA. tuokettles w.th a:reversc. transcriptase enzyme to pmdece a 1abelcdc.DNA ma lc..cttIeand c) detecting the iahekd.cDNIA molecule he bybrtWzint te iab&edcDNA moleecik to a: solid support.
[00071 fnsonid etnbbd tneth b. a stochaitidldbel_based: hvbridijtioit dhai:n reaction methød composing stochasncaUy labeling nile or more nucleic acid mak.c&es with a phirality of hairpinoligonuekotide t4gs. wherein the hairpin otiizOnucleotide tag.compries:n over har.If!; mui fe oflc. Oi 10010 puc.kic. acid moktulc.s act. 1.15 imtiators dr a. hybrid&aUon chain reaction, [00081 At least a poftio.h. of the hdhpin dligotiueleofidc tag utyhybdidize to at leS. a fiOltiOl' o' the one Cu ncn'.' i uc en. add mok uIe Ehe hi1rJn oh"onucli tmde tig may coinpeise an oligodT sequence. The otc iii more nucleic, acid mo lccui,es thay tomirise. one or more adapters. At least a portion of the hairpin ohgonuc..ieotide ta may hybridize to at pottion of the one or more adapters At least one hairpin oligonneleot.ide tag of the phirabty of hairpin. elhtahucleötide. tags may couipnse one or more labels, At least ore hairpin ohgoaue.Ieotid.e tag of time vlurality ot.hau:prn OhUtin1iel0Ot.dC tags may comprise two Oi:1<11010 els.
[00091 Each h:airph oUgonnceetide. taLt efthe i bnlityefhairpin ohgo.ittieieoUde tags Ifl iv comur se one or moc labels F a. Ii bairpm o eo a kouce tag eli hr pturaht at hairpm olitotuôleotide täs ni: eoinp.risctwb:or thoi'e laheis. hi some instanbe:s, the hairpin.
ohaonu.c.Ieoud.etag does tot. comprise a label, tOnal thpknIkMb nc1tägnyomprktcoa\or won in oIigonutStldelaa 5I%jfj: ibo1oSkfidè tagtwt El 3oVbth flt eàth$$ toolu ht ta*flT1*i a4ftflS oli:::ttkt äi4b:: :iwcieotid&n kngth4 TsempSbntbcbápütoSumk4tagoazvbet1sor.
ne twàlstkks è I itjSntt&e.Stijtieotk tswrimie %w o more nuekdthtdS 1* 1entk Th stem pottmn of the bairpin otIgonu6leotide tag can be sent1 a: flute fletffhcha fl lS4e k3cnbe est c ciniei t. can hát:; ji$4 tkgài bna:flOr npSrkjfl I the*ttrnpcn{S øf thi n&It*aø1Sidv,: is 1$, * * 3$, 4O45. 50, 6O 70,84>, 9O 100 o nxc nucleuSes iii ftO*2i The p:podi&Thcñiaganbeonewmmnuc1eotfdn *El4$in1 lè*h, Th$ 1 thMIit øWt*1Ôci m*cleSdtn kbpjh, Th topäoaftheMirpinoligow*4tiJc, g ktjww more øüé In wng4 tS aS $*ht flpsiE4 bfli,e ot i itu&oSb in ante uugleottdes is length The loop portion of the Mfrpixt ohgonucleot*de tag can be eight or ma ntxctcóiti1ength. TI*1oøppSbnorth3⁄4aitpM øligennckofide:ta eanbetox: morn nuckotidea hi IenØh. The k>op portion thho hairpia ollgonuckot4c tag catz be teit or çá$à tt 20; S,$f4O, 45Ô, 4%: 8ö 4t tOóormortnudeoildes inkngth4 Leon) TbSsSøo,4da44 s!=,øg*in S4uei*iifiragi*c, b Mt iicip Snf *,nemeiaS, ntpnIetntflettn anbt r4wm, tr airpM' óiIgrnuteot ragtP ufljij$Sjt:i4 ii,$ 1. tM *.1LniS4ii.
10001 Thibut''rnpSi unittátivtin t$*j MrØ,::':::w4 agion,. Ilito tbSint e:wüqeidSifia! b s*m caihd1mentihe otigwmóiSide tag arsaIpduabSing Sk& SSIth6bligptI$kfl' iio o ctdea tO *61 6lidtpxt 1 at Mat k an itt aenSdt tsáM:suppoa* kfl 4mec:t; Ia it cOütatt $iht!= *ti:ót ApjiUd W.i4i:W anasthtdppaà ía tat tott ss tt alabSd-ntkcui; hrtht t#kiityf Ietotp41tath 2:Sktules:ott difThrettt tlequenceI; the $utafly of oligpnucekotà tagS ton$rtses t& Jea&t 2 (4#flUClQot* agsot#Iaqwnca d Ie1sMvrntka4ecae tiotE ibàilo: tSsm ubtü LsoikI 1*181 4:*$i4 4 :uya plusflty tmóMtuiesiSh. ptwotitSSe;tagnA procbsce a Iab*4-tnokcuk., wbSat the pltmdity of &etutesccqnst at least 2 nekcutes ófdiffaentsequezces a the ph 9 otid, Sgs:4oné : at: hast Labeled-amp Iicou and c) dsothg the abekd-szphcot tO4U9I i!wrdiscjjtS:Srdn Lsakuteoriprbiig4 a piIitytt%psaSaqM:%ags, dfflqtIpn tag e Say ogqn4Otk t* ornpSicsc tatgt spiiiS: á&a udefltL4CrP.P,Iw!4b)*MZy*ø tiM Tn Sbodk ttrSsrot&nnsrjpt i yme in sot txnbodzments thø enzyme is it ligase. h sonic' ernbodimc'nts the enzyme is a polymerase. Lu dhthtenzyaie I ákNase. In sotnean1dhnents thtenaytne is i DNa In nn tnttubka.
tentI birn4 4j$óftfloäidttifl'*:1àá2 uØè4Øt in ia*tb, in tibodimenbUltcuiettifierrcgion is at kast *SnudeS:j kngt& Iniome :4intatg:,tfr!=*is a kt Wt1fl$4, ii kfl r$:t ük.tj get speé.rein thaan:SigodTàquea iname anhra*o*wSSae tag itt&ir conp' a ussj pM St r,oz aos,i*tnwrdi s: m tistpo*isa*mSótiOuppS; Th:04ttt.MqpSIi4sólidtSUppottJfl :fl44.Øj1.jstj4 i*S,1fla a4dreasabkarrtt A rnetdE3& a. !*t 5.
mrn'wnta*pñuted arny or App dM1ornamIyJüc, AMr anzy LgsoineetthodimcSthe jsibeat tøZU Li*ø %j:j::;:&.r 4 Øth*Svt4*Iptter. :h&nStheptirnebindtS Iü *frøSSSt *1* P'thfl: tIbi:I PS*rbU4 sith ott ál%opuptoót4 1$. tsAi
*@sosøueo ipts liI:,fl 1$ n)i1.14 4 é4: 64$ 4t* ttotbftgo&abdjgithybddizatStontSdligo; I 4 si$ke4 t1 1 s,: S: 4L:*p b:S* (S5 mt ss tutcompSa aL' s*me:o,tdSs m:c*1it*i:iónitiI$ø 4.L i bW*1k *WLM:flYt MUü sesbdtSntst tbsJstCyi4e atyt%14t j$26J Insomc cmbodknents the kit turth*r camprhcs a buftbr tt*fli:sonwedinaeSthekit fiitba comprisen t teSi:&noaoJit ua rg z«=q:t a*. : s iw:$* aIityuinckIcaiztrnoIecuie«=The:sy8temrnayeorqpSe4ap1uratkypt ónuioti4e:rt*$4$:;a 4motp 44tSag:at4eSs ppftiøft**Pflutøqdi tk 1 tiSdSorthytottrS an raefo4:&ftcscts t,tfl-Thoresøtbt i:i " c cTh:W1$,, :$: tthW* fiuoresctntaad4r a %bnsotaion or. M Th eant:ttt Inoae:;emboiimcnts the 10031:1 TØjt;ftni:$S, :j s3&enrMther rnp saoq.t:someenbodthenithectff twI Thestm fllsyThstSv camp at njtcrThanae ct nejitkthtz can p..a campS 4lftn ydevlasoth embodSnts the;n sy*vka tapable4rstoi*g 4aSI* $iM$t1 1t:: tW: ** *$. sñtdMOó***& Pts::UftatS writ imcatMh ssk Stoa4orniuc14bJ progra aøs oio: :: ti: Mt*ø::'*: j agion,. In awen bodirne*tLeu* Identffkr tego&is 4 ieask 1Q'aóko8Mt 1egt$.
b s*m caihd1menthe uniciueldiatificrregin cannàt tThnxteSe. :Inome s oiti vS a j:f:..
SbodImettttokiotuoiSde$ &riS onpSsit spSesiat Jtsoit *S:*fl:t*i, a!=a n*iaiu a a': 1:,; JS jasiiks m ioüde&kngt memo atneninhotS: b' a first to: IMti I::nA ía (00$i etabo ut t the a f'sg Wbet*ti a fl yms*nfise chain reactiot AIfluveb am$ifig the *beLe4moiatle may coxtiprS wn4uctancPGF baS anjplMctreat Uf4fle Jah:S-rn*ctt. y ::fljjfijflJ Ia&Ied'jmIetuit Arppftf$4;t&* tiboIe a$f:saMs&iit*&w*d- :nilócule tnay brJ4LSbn Osin tøøitioc RCR)based: flë4hn *Mt j035J An,çlifring The kd-m*cuk may eenprL%4 atnpfit3iingat t4 the label smbin*tS touMi:hi*t* oibo *Isi h i$b.4 :tàreaetióantts lbt 4nthsak w EM I4niIeeuk: In *t 41A*zfts condjaeftflS b thereat En ti s:tOndudtigith$1n I tea MtktM cowprS aiweating a anwesbat primer to tb universal primer bmodmg site of the oligunucLeotki tag. k (3cm P0t 41gM! RTA, or any contWitation thcrtef In some embodiments the method cóifl pñdac*ihg uted& an fiti dd 1Aib* lsonxflctntedPCRanmpf&naturimgd%t Isbekd-Ski*ny!=dutwftc4&uuziIc;bdcd 11* any ptoduet:thcrtot ftta cm&dimS* aSSI sP$b kI røactien fit eonté *ztssiläga;s* tatge. c me iot Suited 1e4td to the unvI:pirbndmpizt:ttb 100311 1:5 nwt dm SS 1 th ani scon4 a reactiOn to dSmStz 11w sequence ofatkaet apoftkin áî the GliQflUO1COti&tagSt kata fthSkttie, anØenttth àreVeEk S: hst i: SbS 16: the HE Tn Soflth t 1tnenSttetthe1abSitiSeSstt yp:i}j( mr:'::s a 4:t fl*::*::::thS*a:*:$:,c, tue scanners tp:somecmbodlmttdenakøukis anueb* j4 tSIi Ia SdSth IkSdirMtcaIi is kPN uè.x wn:: cmbodtmeats t1 nuclck scd in*cule is aq ItMA mekcute. in isomø tnibodfrnints tJ* nnlcvule is a $ptidc In seine embodiments tht peptkk is a po!y$pti4c {*040J In so* embodktants the phnality of melccules Is ftttu s teil 5* some emWd4mc4i1wseppkidmastncc4nsqnw:enbg41mcfln1i.s tMxi aSiit 10 Aik t Some u SUtStaftip1 ftonlst :ejt koi::4a eisnw* a: : :, : a*:sai, nntisJ:&cssih 9tuiIZI Ss.J so*ntgrnb*dLz the it M about $ ce1h In some ewbo4imettts the cell s a siimntUmz celL in a anbodinat4 the àit flnneiLiasommbo44thccel1nwbjcct$uffeTin3tufln4IØease: orIt«=a mSiojenrs *4jcj:jsnr. htsomttnthødãnemts the diseaso or conditS is a patbogeme mfection. In some en*odtnents the dsetia or :nthn&acneLIc41St.. hi rntettiShneit:i, , SIi;ahe4thysubjèct. In, 4oMe nibodiSnts the cell bt dlseaj4ed tat itt sonic enibodiments the diseeted cell is a e*á*sbá iØi. *S' Pa s the S I,is:t4t"' or by stocbastk labelIng, BRIEF:flscflflfl'i$ OF TB" nRAW"GS 190411 The skilled artIsan wilt utstand that the drawings described below are ibr tEft'SS pUrPoSes ant Th& dri"is tit tt}ii SOded tà;Thuit a n&'t any way.
10042) Rcki:t itt si: S bpg t4 øs$* tt If tL 2 sl*owsagná tbr the S ot1bd,'ii hybSisd nidst t FIGS hosvs, n4s Srt tectio*SiàtSln h*{dtzed tnotect [0001 e'4sh,;:'gp*h4dtk'n!=*bsi hyi,,PX'Aóq4ès 100" "1 iia 8 Miiat* ES;the " nx>lecu'k* 10447i EI,M I tI ibed 11)0481 Ffl ltwwa scbmtjçtf seti'n:oYá 1ae1xj,rnóJicz1e,'by'aaaway dttoctor (00491 flCL:$ shonncbemStv of ochSiciabcth!gatz,iundfty utsbxtdis $d Ft.i D': ttprrrniuoà ói%äFPet att $!$ a:gmtcrgpSksequei tO04J ?i&Th aogj pi1As!bStht Ms''t.otiflthci* tO 5 1 no4; tiS show tsSn ttnhe:syntkesis doràiigouScottiàe tags 10041 FiG hA-B (0055,1 na:13: Aádk r1 ts:otLsóLcdIMtrs A)t.abcS Ptttrw*hout generic pruner sequen8e B) Laboted itt wtth unweS tatgd sequence tOOSI flGYl4 Abo tS.RC, ?mbcat t°°ii FI&tbthSMth'".bS usssi:ac:u s: to Itjs s4i t0001 Et<t,flt1entbets.tandar4Inna*4 adgiSIau* 100601 FtC 1* t$gitat microantay probe& -detection using a combmauoai4enc and 1abflaquences tiö ii' :t.$A".'ika4ititaJon iàiiiü1Amoi&SS ky;t*tg'SivSi'.b4A t$2J no:iø r k'A:''á* 1000) Ff0 21 Digitfth*rony** DNA copy t*mber t*4i ELIWS*'my1'SdcMR$M t0th" 2:DgItat xnk'" or S'ttU prnØ wgac& dIgnosis (POD) (acyvkO; (b)cycle 5; (e) cyele 10; (d) cycle 1$ t,0ft61 nthtüt ibrâtgk eé1t:prc4rnpfSSgsS 1*71 flG4.D*iStâi*"ty'*' :Sz $at aài" flóMy ii' ntjoISaó"-ijrny fl 1l,R S W4tthA:P$_øP, tsiS'iS',,,:$.DA t%'l FIG461abe1m*wnt 4:a'; M701 ez'ssis a n'a>k'3t-otu$tpg øPPQ4fl' enipIitidaSo: 1*4.1 ntLgS A:n:'m,tkt'tcefls reon &"t'4"4't sM tGGfll na;29Lt,r'lifi4o'ith*' t$j ittcl o IA: iñ1i& 11*141 PIGS1L&*lthfllat;prlSng (00151 [00161 and **IóS (oonJ rti:ss:*ngacLdsaos toOt I %i9( na)&A-Ei Shows tht scatter pkis*fl'<Sis thrtiit bn4rp4tSeS sôith Ø&4tMt*$4fkiin*j*, JS& (01 1 $bc..... 4 CopitMionyaØ kth iSibn:xwdS* wkhS4 AibfSsEianamy&.RtA RflI cL3 Stt Sf he Mth4411!*ion. *ctøTL *ó*fl4 as*., jjid6tons oL:humáThtRNA S mnwreWPIfl txpnSn (9082J HG 39A-R Showstb scarpbsofrt'suks fbrtbe titration apcrhuent st aaMknLGf ham UvcrRA nmzreClAPbS:eression t0O81:i bAwthóSttaph b t S iu*speñtt wIth Sal dUutrnns of bwnan IivtrRNA to measure CIAPDB eptvst (*:.1 flO14iA-b gncs (0851 nt4ZsJiwmMsb&fOtvaJ!dài:k;otkAnunyctssJbyagIt$t joeiij:cY4. *tss otute:stioar *wii 4ti45øtfti. 4 tsfrSL* DETAILED DEStR1fl1ON OF ThE It4VENT1O?6 to:,, aiias' ,ss' n S e'i a", ottb nta WhiWthe invontiàirwl'lth $ti" P.rS$$*4'4:!ø li$:,.th',,'4nstiPP to t''':', irt "04t toniw,3'4h invtnSn is intcthf cci to taMtiVeoda&Md'ajusetWh1h nsbg bà1u&iàhin t4$'it attiscope otthc ivouth*1 [00881 Th,:ifr$Sfl!,1' *o**S4 $ltø fl$"a apØntionsaSt oSretrcn'sfl isi"wn't'seotSaitc fl*"rciwkn pdent, appIkttio; eroflter rcftreoce sack as a pridttd puWinton. is eitcd or repeated below4 iLsbouI4 be undcntoq6 that it is incorporated by efErnctin,*Sfretflnu posandpatuoIartAc:pmpositiotzSWmcitS..
iSi Atnnthwt4ual t tli d toaMimait StthayMfl:othathi%t 4ftflfl: :a,*:api**. bs:t iwLds&:wa: 10090! Tb i1tdikt *b!$ b:SPWWMbi:fr*sI4w :angttnnat. Jtshodndtk4Pth4t:f*tgCSst4SdY*t snvSmcSbraáuitnotbecaørue4 arIMSia ara iS** Sici:4it! t: 1S1*é4 töhâE iiiceflydiSk atpssibk thhrat swftkdh4uatrniShzcwwMt tSraag Pare*atnpk, d rtohtan$es1aast1b1 I t*6tho4d be dóMtdered to hay piiSy 4SSbrgcnuth ast*, ttotfrórn I to4ko;flo £ *om:Zio 4, fitrn tot Eo&;t* 6zS well brt:i, 1,2.3.4,5, as)d & fls applies jerd1ess otttie breadth f the rangp [0091J Disetosed here hi axe nt}ads. IS, and sy*,s fot 4e1ectiu iwdM qntfl&iflØ molecules in a sample. In some hstaace n*tbpdh+ kns, and systems St individualLy cu4'::nxs1ucs iuøvsanpk ar pmvido& AlSna$vely. mothodi ki.aS stemstw doteSifdng:ih:kt1dwef*ft Qfltflt geno:prud t os et En saMstanc tb&tSl4' . .:t:oM: aiSft4aj tø aa spe$fictgj I4U4: *ep t,,..fl,y t *flat34fl: iOo1á WS$. S taflthe:nto1tcuS &ini&ttm iión Ma r4unàjofrtcmpdSg I %at 4pa' at teüti jiitbfl thdüWM *:SW1ó*4 c4 4nt4t*fltpmd*4 eabe*SSnetby'dSoctb%g an4'tn'qIt4ngat lbMtapotEiSot th Ialxtd nkwEe (e,g, uiziquA juttetlo; oligotrnelcotid4 tSg% molccuk). 7k absolute hydet4hg*Ooi :óiiaaikii&nagsotS WtSulcrbeitumberoftquejuncioifl:thc labt tVjs t°°' 4tcbsctbSn bkrno#óraVQr q$ntØg ottontre iioStn A auejttS4 S IntL t4:s iissci ofllt:14, 1ijuøi4t *i,O4O4Y W Sfrg pnnibcr bindMg sit* (1401), w*zt dentifitrcefln, (1403) and a tatp* spsifi regina Aapsw;swp**!ri4;:S, óflgoxu4idetag{I4Q4):usy Sapthul *. cpy efthetergtmoiecük(i4GS):can 1M $ynthL$tZcdt:pntneXtetthfwflwflQfrntcvSsG (g.,bNA1rncrutupm4ucean an$k6i.(14ö*tho a iitohf140)n c*pge a u ii eSt prst b tht (IM:iJ, :%Mri 14 4W pnnitr(1407)cannueal totthc ui step4fFc1 t4i$:nuc:: I:% :b lug iàø.
azuplin(t44a The S.p1iSi::fl4S cancomprista:cqpyofthtiargàta*cuk x4 v1S nj&tIsug 6i1 (i4VS sesmpietrtenttt i4j4%, 1:408) can ttt* i for subsequert ai$ffiéathni** a fnartpt (14$y itS4**S!*o jj4 A tPvI:: !4i$ØØ$ 4tt cgca *s pUcun::cGmpm stkun*qu enflng (1402).
Bymeor.* th i idonioi:gkiSUth amlia that *1? atlaS with cacflargct molecule Thc absoSt PCR method can be uscd for sü$uenttIsisMwtapt utea(Sip*5oIEIG. t4). Pot nampkthe ampikons IPDOthLCS ythe4 óhtvc tR:xflett*4 tai t uscó tt4t&tandorqu f;flntofl1Ubtt: t!=:J&.a 1e*WcisdM:ith*tf!1* gsoth$g$*cI$d4 tags; (*1 4 SktIkb4 t4ói Thmetht **ui$ in s** b 4W Ed tdp: id*tik* the moletuki3 an be fowt& for txamp1c In U.S. Stat Numbors 12/969,581 ant L3I327.$2 piurà1kot :óiiamikii&nags tontwornnuJetTheflwiIityo1nuc1wtttagnre a om. qpn::4p$*p J gip 1a*4 as idy4ianokqu1IdSasa indiSua.ptbøbi1y:*tattaoMn&i aiaet 311 $Wiity*tah:SMttuairnohtuk to LaboSMtaeMng t4 4 iaMdop, thpkøS tflë Monlingi iRaitS, thpb&aflo1thc'tnàiueiS ann4Ii fiSg *yo$tg S' sMSft cikWat,sto csthnate Mtmhi. leçtkt *ewk à .same qirciuuflanca tk :probabflity dnudxtg can Sn*npu1aS y. M exapkc1bththge *hdietiin prep S. tEat nin: : otSt' Sthái#iSySS tag w'th aSSMI molecule ThSgosuttMfidt fags caA as bt sd in nwnbei'stU alter U*prbthiMy that Fotecplnne oicrçvxèdtsflnt at lastonebà'iding.pnet ti%l A hafrauutAi*aA o4adeSktøsoumSèSè toezi t: j: tStbd, iniflüdi g,bt not liatd totybridSthn oc:n :*:q4 n s*tnm: :a*$ .: 4:cP:Wat4ot $p$Ø Sq 1kw tagi1:sp*ctwgk, qan:qu CflcqflactthM. is Lemntnyfløk,t ptth,, at :4 IS tatg4 iØi&' tegiodcat dSttothkcttleiSS, rsdMtw*b.ø [0098J Attachment oUthe ohgoiwtStfdc u* to a iSccatc aotcur by hgattn ti4tpchthUeS:cø**flt4sui*pi4 iLta$ anjSjp4, NAli hatNheZ t*Tkt LPP4AligaseW, sn414ji4A ligeLi4tntntiWucan thmS&A$A: I aseS swdisetA thkUgat:atiA:SA Loosi MtbSsstfiiiSwt êscribed4tr eseS:M unbwoká M (( fl Sf1 the ir1o* ESitbtihstai hota4iotSy;;S*çr&cs:fri $flS1,, 434n*k psing N DNA LjjSfWhè :41fØ:tL! opk I iódn t*N t i a itSi DNA or tWA wttb blunt aw sticky ends3 Taq DNA Lzgase which atatyzn the fi,tmation of ap ospbdbsri 6 Seen lead 3thydi. : yUeSni ofëwo adjatcttt oigonucleotida 44th an hybridized to a tcntpletnentary target DNA, E colt Dt4A pSsptenS MyMxyt ss: thctWA tobSve;ç:S t4ktA of aS' S!=.wp**ts.,** s id döflttb a 3' Ihgd$sykcmnaiodnudàk pit khcbnttpdr4fr:pboiphodiiasic zbstrates ngio*trS*d*M StM *wcua 4awSeosije; *t at n: u* r*n!1: DNAfl1rI**4.
wtthors tgnj an4onStprkvt*A3gatwetahptøtsttme oi ck*fth iiéa6ót 1001001 ii:sontswabTh entoft gonSeoSitganatk lathe I3 I.flc3 cu1c Fy both cnd OJd.. }gonuckotiJ ta an be hybr idiicd mJ hgnd to one (*t rPbre erxthoftIie. mokcult. in Stme:... attachfltit M both oftlie (3fttOflhi' ké.tid t.a t. boUt tndo 1th4. :noIe:cik flSt hS tfl the fOtaucit ofa. ccuiarzed.
1,eIcd.mkcuk;. Both ends oFthc oligonu:ckotide. ta can Iso bc c< uitcbcd to th.amc**cnd 9fthe rñokcu.k* the 5 n4 OttbC tag is ligted. to the 3 n.d of *thc. mokcuk. and the$' cnd ofihc ohonucftoüde tag s hybridized to the 3end of the rno.kcrde. resulting in a labe,t1rno1eccte web a ban. structure at one end. In sotne stafli the e Ieonaciconde ttn is attached to the niid.dlc of the m0ieedC.
[001011 lii sonic instances, atthchmcnt of the d onudeotide tag to the molecule c)nltthes the use of one ot iore.ad4ptör /-Vlaptors ca c uprise a tatt.pecific rego.n on one end, wthieh, allOws the attachnwnt of the adaptor to the molecule, lUid: an oiuwnucleoude tag specific: regionon the.o.th.cr end. which allows attachncnt of the.o1ionuciwtide; tag to the adaptot \ciapuu' &n he alLehecl to thc nloleethe aM at aiftontleode,v rwthoc inclwlmg, bn.t not litufted. o. irybridization and'or Ruatiora, O0i 0I Methods tbr ligating adaptorsto thtgntents of.nucleic acid arc well knowu, Adaptors may he doubic.stranded, sinie.stranded. at partialicsingk>stranded. In preferred aspects adaptors arc thrined front two o»=.igorw LotirLa that hate a region of c.omptemenhthtv, for ekampk. about 10 o 30, or about 15 to 40 hases.Ofpcrfrct.
compleemiosrity. .so.thnf when. the two obgonudethkks. are *hsrbri diStxl togOib.Oi ihe Ibrm a.
Uut Mc uanded cuon Opt2ona.Uv, citici or)Ot i ot the d onuciootce in iv a egon that is not caruplunientary to the other Oijgonueieotide.anri torins a. single. stranded overhanu at one or both etid of the adaptor. Sirigiestrandcd overhangs may: prefibrabl by about ito abodt$ bases ahd:thot *efeñibly about 2. to abodt 4. The overhang may be conkjileth.entaiy to the oserhane.createct by cleavage with. a. .ntstr cU.n enzyme to.tacuhta:te stiek:yend" hasuor Ad i ltor\ mae a luth otici features, suc.h pi m. bndeig Uts a. U re' i eflor sites. hi some aspects tire restriction site may be for a Te H S restriction enzyme or another enzyme. tha.t cuts outsideof its recognition sequence, such as taL.1 k. .11/cl 2001. 3 2i,4);C4Lô98 and.S 5Ji.0.000 which is-incorporated herein by refere'niee intts.enthsrty).
[0Ot03J Thee ligonucteetide. ran.cair be attached any region of a. floleCtik\ For y:rr.pie, the oiitomtc4eot.ith. can he attached tt te 5' o 3' end of &.poNtiue.lebtide (., DNA RN.4).
es rm,k, the tiigctpcutL reior u"the ol aonusle't tIc tag con pros a scquene thu n ceniniomentars to a.cqueice tn he regnn a. the molecule ihe tar t-spcffic Nun of the oheoirujeottae tag can ako eompae a cqucnce that i', eomplenientarv to a sequence re 1.4 *th&SreghnoflbeSkeukt]isomtMances,tealigeiuickótidetag4attadsdare w#i *s M *ióh$ó WfrnètP$fl è àt**i Ap iaItcrnthMiy bft::t$ :*sOi eas attaEhed in the.d1 wSoüdc. hi a enmpji igom Sit tag tOol 04 11. Reverse ansafpSn 1001051 In soP* tnsLaices the methods disk3se4 herS compri'id attadbStit ot'an flgeS:oti4iiig p S$A rn 4itbtdasA i*,m is stttau fürthet Go e.rGvetttanscflpttOn. ofthe kd-RNAto ltàleto sd*:1* W*b*èL: J*i: i*:4t4!:$PcS.
iLowdteSdO aó*s a:pSnthrttenxse iratS.n intk. flu: Jiown in Ek I, Stçps 1A-R an offjonuetcetide tag compriaag an oligodT st4umce ltflridizesto poy talE afAwrnSN4molecS Thokc 41 postiaczft àaStMeansan ptwEbt a4syuitSt bkAhcM fOulUóJ hi wnic instances the labósd eDNA molecule cat beused as a molecule fbi t a*dSib gi*cfla the 4ektcbNkcnha&fktota&iagstom ft* cD et trjaie!e pgqi dbirnS, dia etc eAflO rttsZSocbUttnttt*tbth oftLi tat s:L a$iPó 6t1 kc$$ ::sto toftiO7j moletule to pmduce a kthele4-RNA molecuk Reverse transcription of the labekd-R}4A nItuloSip 4*fr d4ftkiuftrs:jn*piist: U41*o hisPtjia tho sflracptionpnrnnanohp tmert ta dorn:hnarnscteotidepimecot a i2_1?:fl**t*:**th S W tt gcnospo(AftWt It tW Pa4ofniwSliu stskRMdorn leó1eitidbprizins OOiO8 1n:srne Aabeied-RNA molecule to producc multk,lc btbcted-c DNA nxoiecut%. Ths methods chiwlo3cd ter$ft;canccmip$secGn4uctingj:Io$itabouj 1, Z 3 456t:&t 1Ø,li$2, i3, t4i$ is 16,. II.18, 1 9 (): 20 reverse transcn.pnon. roactions. The ifletihOd can co.nip*ri c.ondueiiklg a.L eat about, 30 Th O b MJ r,) & 1() O, S YO 95, or 100 iecrc tdiJ*Lc v jE)tkUi fet.ins.
OO1O * lilt Amplification of labeled rneiecuIei totFtloI Ta)flCthOd' ci sdosed hrn wrs coilpn'ic nip1 tkat on i nc iabehd molecules tO pioduec labeled. ampteons. .Ampliñeaiion of the abcied molecides can comprise PCR.'bastd. methods or.non-PCR based methods. ATnplitThatiot of the. tabeled rtxi tecules i:nay compn.se exponcutail amp htu.attor;. of the labeled me keules. A.mhficatim of the labckcl ruo.lectdes may comprise linear ant1 ificatoe ofthe labeled mokctdes.
III ft moe nhL. ILes, a'nph'tcatvn nfthe Rbe tJ mokcLks en ipn'e oriPt Ft based method.. 1E1⁄2.amples of noni'CR based methods include, but are not limited. to. multiple u o1acemcm ar qi'on MDA) [n mLrlpowoicdiated anr'htTk,ttiou. Pd A mu.
acid scqiencehacd amp f3cauon N \SBA t tiand dtpIacement amplttwation Sl) \). teal-time SDA,, rdfl in.:cjcc]c amplification, or cirdeto-circjo amplification.
{GO1 121 Amphf1c4no oftht. Lix Lxi mokcuhe ma. compne h b Jtz dine chairs e< Uo (HCR)ixised methods (Dirks and Pkrce, fW148, 2.004; Zhang et at, .4 ca/ (hem.. 0 12). LiCk based: methods mav comprise DNAba:cd 11CR.. IHCRinised methods may comprise one or nio e isheled nro1re I It one oi norc labeled piobes m.Omf hC OUC 01 mm e oh.c:ontaticcttideta.gs chwloscd.heret.
I 3 C sothe istsUtnCes, the ttiethods disclosed heteht tirthet' ni:r cobduethig a..
po rne ase. han react sin c'n I he ibk' d-mo1⁄4x L ( a Uhe d-RN A, labe edD\ A, i,a.behdcflN A) l.a pro duee a ldhetedamplico.n, The labclcdamplicon can he. douhldstrandc.d tholedu.le, The douhie-&raridcd rnolecdle can. coftwriae a double-stranded RNA mokOule, a aouhie-stra.nded DNA molecule,. or a. .RNA mulecu ic hyhriclizc..d. to a DNA moLe inc. ne or both o tia. ,nancl, of the euubk-sn meLd roku.lc.an Lomprise the o lgoct a kotick ag )\hemnativelythe.labek.d-ampli:c,c:n is a sh.glesttanded r.nolecuie. Theine-stranded mokoale can cemposeUNA. RNA or a em hination thereof Thcimtleic acidsof the present lflVCfltiOfl can c.onlpnse synthetic or altered. nucleic acids, fftJ I.4J The pol.ymrasc chain reaction can be pertbrmcd by methods.sueb as PCR.,.HD-PCR.. Neti: Lion PCR. tgu.al RTA, ot atly coatbi.natctn th.eretM; : Add.it.iorthl PCR: ttietbocs mabi.de. but atesxt hmited..to, :a le4pec.ific. .PC4t, Mu PCR. acscmHv.P.('R asy.mmctri.c PC R dropet PCEk, emo siem PUt heR.is' dependent aniT)l heaon HDA not sLr PCR.
nsc's PC ft lin u-ahei-tho-eponential (1 Al D-PCR long PC ft mu] ll*\PCR nested PCR. hemni-*nested..PCR, quantitative PCR, R.1-PCR, real time PC. smlc cell PCR, and *wuthdâwn JPtit jGiij *tflttiftc tis tk wi:t oe d*inntimttoxflprsig:.tMsimflt*UaiiniaL cs t:t#' at hTSM oh dmoiuktLiSi.aniplicor. thenStkot disipsedL#éi can fIthba c*inprs.
aaeMiaasecw4 ta,cipeeih ptE ist itdsiAit tOOt:UJ iii w pitt II#kOflSt1YC So herS tan comptS cenducttng4tieast*bmu t,2,3,45,6, 7, *9j 10,I1 12. 1 I4 1. 14,17, 8,19,or2B a$cannaction; Aterns%vQLy di U thpdcqnrisc%con44haLkas èbSfl 3Q sO1 i0?& scc:sot4cso aüiP&&tovtt±i*flS [%i%t7f: Qtt *iis $1' M4:: :, I:. 1*44, 4h4:!Ig:dfl $ti:,(LC:$ tIE Wit MdLWt1ic, tanite thjà,42*J, Jon (19S8) and Sau4ngsrct at. Geneati7 (1*)),tnut'criptiGnampU&ation (twøket al, Psv $*z!=4am&.: tS;$&21t a(à1in at? Not Atatj tkt WA I*741)ikknd 4':$.
tess ne Si pó1yrncvasOi (tnt:) <Jt ?atStN,.
4,43775\ arbItrarily primed polymerase than teacuon (AP-PCR) (US Patent No. MW.4 $4tZØ) iliig iS:k *$siC *0)44W mjjk Pks4;)4,;?$fl 32M41 (19 5$w3LinVa&, i.s. Client i ItS' (t99)) aMtL&Pst S,64$,245 strand dIsplacement amplification (see Lasken and EghoIrn flnds BSathnoi 24o:n(l23:sc.: Ra4aétat Q meEt dJMayi4($)614;DenigL hyjc$'itt tad Sc: U S A. 2002; 99(8)12614, Wallctr s at 1992, $nckie Acids Res *7). 16*1$ ft*2 ad i,",, :e/:QtN k':4éJ;kES; Z4flM:fl,QbiSbJkSMj S't'11t*d1uiPt1' (t4AiSA). (& US.P$Ottt Nos444e9&t8*S45544n7n44jD63j6Qi3:eaa*fwiuchis iptcdIkerà y: Sticflsn atig:eth4iihai may bead sr nridn:LS flwsW6i38 4 L4it7sn4 USS No1 0143599 moli ofwbioft iqcosad hentiq by referea DNA ay also be *mp4Wed Want and Vogtistem. NAR (19*9)17:3645-53 Othcx avaibbk nstbo& of aitfies su4t;:t bitanced tot aiii (®2), Nwsseth,sNL* pS364)6 tflt)! ti aIsobuscd.
Ioiiiii Motecui&Sc.tsk jntS tMf th 0 1T:4&st cempkmentazy to rcgwis that flank the iegkm to be mp1ified The gap am it ckscd by c4 a:ft $sS:::pfthnflm:c: iMtPprob'n:uftheentt:ibnnathsxdéftcti. 1:bcSt*ékcanbeaniiliti Hatdetla iz4 t%*5si$à.
(00119J.
jwitncrs. FIG. 9 ghowg ti eMmplary flrward tm4 revn primers The trwlrd tSer (901) my enpSca:a wthnflCWsunct(9Q2) iruasditt :_N4tThtoM.P1_at (P$ utaflpri * target scttt usuor: c::, j:':s,i' ;M4s ot*tioi:ttbmThs:A.
on anser tb*&thpthequett Othc&' prImer deetgns nay ako be uS. or pnmen may be sclecte4 by ?C without the ad4 of cnhpu$rpr,,aThecnanyopsn:ailthInfl;ftog!arnIataibrthepraer M%gnsniot ç Ii M:'Ssuntmaytontdudth& tw*nwtlflked;toQ oligo melting temperature, length. CC conreg, 3' tabiIfty4 Simatod secondary strucuwe, the resèar4th'&$OtS 4Sr' tbswi uso copieoMe se S,; trnpctatwtjht likm""orpria4'r, cnttwo prknoaMThpak1 *4s:':,**',,,",' IPth* ti9t4,,$$i4!ü teg$flS f lnteresttfle aw1,,' t$'i'i. W. Sequendng 104122] biorna'4idió nitti'dá 4'o aedbmmfisedenMning'tbe.
"setzeflce ofliiSbimo1eàleorBnypmt*1tr(e4fistledornibèS sso::k4 an cornpSc eductii' ascqpaSg rt'sabnio dèthn*the tscqusc ofat,kt apethn of the t1i''*1Skic t*kat ten to tSlwS*. znoieaao sto*qtSrsnt S,,'p4L. eé iifl*OnnMt 1 Qray fl 2!*tbetecd fl' *4$: fkS*'1$' oj%t flkehntM*4untthSS ieoa t::"d*tt"s'tsii S"'* W*4w4d* botà4b ó: HekoscopeM süigie tmkat scqucncmg, Manopore DNMequencutg Lymi Theapcutic& is \1a,% c y P4r4UU Signait Scqucning ( MPSS 5+ pyrosequnt:J Sink \1occ e ttflt2 (RN \P epenuiit Ukinna i)ovU equccig SOL iD equcicmg Ion 1oiicnt nfl s3TTI'OfldUt J'qiP 11t Sthvk \iOkCLk sIR P f v1) cquruct P13h5 ly ecicflc n DNA. nan.. cybadi sequencng. and VisIGen Bio t.chnoicks approach. :tUteru:tve1y deteitrijoinA tbe*sequeñce of the iabeled-'moidcttic or any.roduct therdoibanuse equenci:n.g ta ttym' inehithnn, but not bnnteL to (kiome Ana ci \, H S.q anti \licq filet ed li3 Ohio PcI Sin?)e Mr ki ute sea) Tne (SMRI te bno o r sot I Oh' Pacligi RS ofuzi ul Pi dh I n,ctcnce, ( <flu nu.u and UK Sn) >t i ltu. Sii L Moknit eueueint (tS M5t) tcchn<kry such the ì{eliSetYp&M Seluencer offrred by t-lcIiccs Lire.
(Caathridg, MA).
{00123J Th sonic. instances, deermining the sequence of the irheie&moie.c:uk 0:1 any product thereof ca prises p.airethend seqpenci.ng nanopore.sequ.cncing. high4hroughput sepeio:h.g, sh.&gn seqiflcitg, dyedrthhrator sequmçfl tmrftip.Iprinier DNA.
\cqucneunL 1 P1 er Jkmt!, Sauci dn (;u ig ig \la'cuTr CiRidt cquunc ITQ, po'cruencrng two Mngk inokeu.c <cqucncmg, or ai eomhiaLon th:ieot. AItewarRch.
the sequence ofihe 1a1e1erhmoLecuIe or any product thereof can be det.eimincd by election inicmscciny or a chemica ksôns:itive field efflict: transistor (ch.etnFET) array.
[00.1241 i:nanoher cxampk. detcrth fib the se4uehee of ahe1edsoioic.cuIescr th p.roriuctthcu'cot comprises. RbTASeq or bucro)NA. iiiCT1Ctflr: Afteruia.ti&y the sCqti&nce oi iaheied-niokeulesot inyi roducts theretf cOPrpriset4 ptoteii sequeceing lc<. hi fiLL S S icn io, F Unian JegnicKton u p d n ns flngcupi wt'<g, nr p&Uuo'rK'Uy, Q protease digestion; 1001251 The sequendn' reattion mjfi fit iertajn enibodikrueths., occur or a solid, or sxnh solid mrporL ti a a. un an emul.itm on uu lace on a licad in lu op. n r coinu to!L4w it J$ution o. in cm. 01 n1oR. pb'sal enarate \o unic' 1001261 cq <ereir rnsy comprvc eqrentu <t 1eat abom 10 20, U 40 50 (SQ O,SQ 90, 100 or more nuckotidc,r or base pairs or the)abeled.. molecifie. In so instances sequ.ncing comprises sequenciLig at l)rast abOut. 200, 300400. 500, 600.70th 800., 900< 1000 <y inure nueleotides or base rairs oftbe htheted molectile, In other tustanees, sequencino CO1UPU'US sqwncng it kat about 1500. 000, 300fl, 000. ,ooo tJ100 7000 X000 9,000; or 10.000 or more nucleotides or ha,e pofr oflh.1be.icd b)cfiecule [001211 eu enctrg mw xummc <ft e"t tint 200 00, 400, 500., (SQO IQQ, t<00, 900, 0th) ci nuue scqucncm it> 0, pc u.n In some unstanec, seqne'ic P eompi ceccnc ug at least about 150.0; 2M00; 1L000 4,000; 5<000; 6.000; LOGO; MOO; th000;or 10,000 or more equuzS1gtedsper nm iS; tpsS:Maod tOOt$) T$e;ta*:flS* 44j bbkd.
*Fft iecuLc IiibcLed- :um$tsons:4ãt Wi4$iø P W**$ i: $4t è :$ Ii4n:kQ1t u0,c its Ip jow:1 s* :.tt:.t1oj:is SkM h I*W4:ók IEabcSaptS'*kk a:ikath*1o n& :;ttabIt4bci cojpS mOkGttté. The ttho&$ 4scbS heftw caji further tottpfise d*azng 1k detoetabk-13861 wuJqgSde.iwS:tS jbnQft or any pd:1hçrcof Sg S k os4teSatu jttS i d:staoukj 4:kio. af:ffljse $ kbflhoIià#teft4j:AJauk,, 4ét*i,4 conJAn,S S:I oXimuetStkiug,a crnpfr rottbesnleS; o*snMuáS ft$fl* DtigtheIabI4-piotcu1cspr an.y prndüct*::therScansompS tan it::E1 et4M th&k 4Shcu1n art xirta an Akernubwty,, detccthn nitbe labeled-inokoiles or any productb therfconrises ozc at (1Lt$fl :thtcj ku d Mca1tonjugMcd itodn 1ctSt eontdsc dóSngui It4$!jdØ tpi ii t't Fbi SI':! *I$$ 4 W' II htt*th sajasIngE tout a orijonøEeøé ab$ed ntlcc pie thereby dLttngtaók 1i.be1ed 104132] Lii:rnØ M t*d*tSotdié tab noiccuSanWer * them thtc h*Siosttiwo,: :::s.1ssSss::. 4c1ót & ki tlsn*th 4.Ss 4bte6tbtofs thiur:t4 D ii tb1ab 10 dyØucadàooFóM mpttco1S;thoàeaj4eS i4fti n:7is&t::1S 4.*rs.cncoki$1.ht Array imaging Readcr (CLAJRA ESEQuant l4erat Pkw Immunoasay Røadcv, SpcctraM*x ::Mopcat:ssntt 3M, %ySSMax i ysáMax, fl atuEEMax.
$r1: Manrna]rnethodsfused4loneorwMoInblnaixrn wihotnthGdstdetttt1he.
t UMtftaSk decbtft1wt thsth. u*:iete&t Ic is W1phost*nØLsst:nataaaatq : :frSkd* *Iea* dotS ó4 $tottatntd$ctr, HtJt*rdàS:itnyticSs, 4niwty4 soti a.*: s tt.
M:Th4:MaZAiia th:, th tStorcS bta n*warray derSor WzWiS'itg exrnptcfthicroarrsr4ottttorsiiSut mky4icctr tN Swan:yttI1or&au4 prprnkmarray4nL t1101341 Thmninhi*a tuots:rSS is ueO ft:ntrays*roHwr Strttfl$ Mb ps:On *ihuiitropkttct tnsome ustauces, the fluorcsccnce n*dera* &cnsovation Fluorescence Array Imaging Reader tAiR;.. emwence ri jr!*vreaceuce mk*rcad,aSa tGtrninLXfl F senteR*kLtcteadtt Gnniüi tihttces rader Fu wnenb44 Fluoroskat fItter based fluoresoe'nce nnptate re*der, PfIERAnaxr rc*ç PWAtn óSth on: PbMtbs#t Pht4& iwuj:. :Øt$iü pftki, iW :somprSs jJ tin nkropLe. ,; re*tr; in sazhsa,a:tiinnkmpSneadr it an: xMarP' nncroplatc absorbante pectrepho ottcr iMatk nncmptato absorbance radIr, Thi$pfràø MuIeo'it ót EóViIii tttftitb IiiMt SI V1crcLt XMuIU$*l bt;rts ii n, $p axSa4n. SiMx M5ctraMaM5 SrcbtMax M4. SpoftáMtMZ, spø .:. 2 FiltprMaFa1tdtPskafl Aset flL Micpkt Sser:andtunSomeieçAuomskasAacent MS$ait PAioremtie tatni o6n Asednt MkroS litiinornetet MiMkatE Mi*p We n:*frtMiistsrc. p' O4MèiiCUMftS$ pis*reaS d bsorbattct4 flrttc4huiàj s øfl::z!ass embo4in. the:rnkrppb*t reader detf:4ytniç JiSscaftering The thieroptsveador; can ikt so me. Saws deS static tiiii. saitcrhg. in bstanadetcctiöno.tthe, t ith'eflt* use: ótMSISgotIE a44em:::::1w j..::::::v.L:::4.:j ti BioRad MkripS. mag:i4ysSfl..
fOOl3dj Detection at labeled-mo Iccutes and/or pwducts thereof can conçrzse die use ofa Iáznbmóter.:Ewrths::oflnninomSi, SMaS it$pSr*á 4i:jjjg Jur H nkt n1nometctt 1oiS Mukf with litstThet software, OloMaxE.Mülts Jr siugle tube rSLS$t reader WM[star flThISADEk Heth rnSLAJMlumStátt*ndtAbEP. MX tOO*31J 1n:irnin4n's 4ctIon of tbc' hbeied kcukwaodAu:labekd-unplice, StoftttScwihehtd&:flatid sudiatThuSe poWde$ ItL:aMUtFi4ss4tem4 Scanners can include mkropt scanners *uchas lbst Mnyft AlmyPv&tM nüáoarmy :rnicroptt sqam banGi}stancet, zannei in P4lmagerTh(PM1) qiaemptnidid by BksS touui that ntas*re thc maaatacbargc a of dwged poxthzM4 e mass spectwmcny. In sonic emb&dinins the MsMocharge ratb of tharged jsrd&a l& tflC*sutc& to cottatton with sepS: :Ss hs* Si sá :te*4 SI, in tsii io4ac S ágepsi'k. thcaaurentcS: M SOLtE *s:s:,n:w::ss :tñwolM or mani$$atd lii: the thflhrary j90139J DetectiAn of the Labtkd-molecule or any p oductstheitofdon!prL4es the use of * a1làj,. iSti*b:ig. Fir Sin$, ,,.aj:lSd:r tiénlj (thg, labtàd- or&eátyto:sniflpattióles and hybt'thcd tothearrty; Th:i! Poit$$ t 4:hSss* to micft r ttge Thr$S 4Std sL.: llgr is S, attpHS:sirtaa tii 1401 Astmw' M tt ww t sni4iS là ataobStpvts b* :whcS:'*4ø* StS* jt$Ø$*h$i$ :4 b uS to 4 the1lMauS*om*rth1 *orthpt**ttobn *ligk*r&nwniatetMEcan be used to Ltek uadS' lIg* sc erróflectiqn. The light *S$iag matcr eaaS a %ed akthrg or oll*niatetigl Iwoome hitneee dttcttientxt tho St aara:iiAa ij: Ooutj.
:Oflfrdjedjii Fqa st* t*4s a (33401) isstóthia Ii 4Wieted wiëh tpknl iyotolionuc1eoxidethg,c (iU) olignatteotlde tssjs (3330) conipriseauniqie itbnttftet rO'en (3310) and an adapter region ntt oftg *i&g 3Sb)to4neor n rttnt;ófthenuddWfda,o4iSk 34c) pto4ucb one Or more lebekd-molecule* (3343) The One or more lAbeLed molecules can bet u with aphanIJyotHCR pt:bt:(3$).1h. plurality otHtpSes(3 may co_t a1ápiat1eeu1S wilin ontg andonai ntot abei (6t St piit8cr$* Q3$i) comprise a stem (3310,3384), The scqiacnce ofihe stan (33* 3i80) tmy be con meatS least a portk n4Uhe oswoti4eS.t:equencciffie*m (3SOi0)Wco*krzMtothg2U)cptithnag The adApter regina (2320) ctf the ohgoauñeotide may act as an initiator for a hybdthzation :thtreettAs t *iiS1C.3ltstsrnQ3ictS Mt* Mçc3$) to the adapter reajnz (32O) of the kbeled ntletuk (3345). Hybridiatton of the stem 0370) ó(S$.CRpr* (3.3$*:* j:3$)oijw:te* fl4& sit :*:øf4l* w*::*Q3*Dk:ie:$ 1t1* tiöttifä1*LØd iS The tSe Hckpmbe:canmettaa nan sübut.hftidinofanotSIHckptt1t Mischprubq can bØ:idi tj:tlieIjeaft' I CRrokthàt t byb'd'ed io;tt lA iótt4à, rt$kdthj ttrca&atSoft an&itRpmSandS SmnS:niIeS&aiig s*b:4Cs.vThb4iSd:I!= scø pbna fl S Miathtot i anoSSizath)jb& Th:p cc*:c repetedmukp taniefl:pr*dticc.* Rca pI $2S$;bbt1t036 n*O5otS Ifflpb* qii èiiaW d*bt4the W.idIt1! Tb *1fl1 $!1t$4iy type otlaMil (tg. tbiorpbore, vhromoplwFwanaU m*cul nanoparttlc bapten1 enzyntc MbcS(33t1}athd33Mma'çqiisqfr8gticntiöt*sig10labcL The labeL, (3360,3390) may geneate a deter*abte signatwhen t$y are in ckae promniity When ih I K It i t*c 1% 3 ItaItpit'. the LN I 33U id O\ iii N. tou Cu dk d to j'itice de.t.ectabi.thLnaI,. When Jie.1-ICR Ørtthe i* Iin5aiid atid.niuUiph liierizd HC:R probes art Ebti.1 jzed tü.gethr t}k tbeI. (3.360. 3:39c) be. ii dOst pogh. tø nc*it A dctectah e \IgnaI I ot c'anipe ci Ikit rc>1x ( 35O) may otrpns: ho VRflC moetic 43ck 36O, 13101 \hC1fl4UWk' thc 14b* iis h nanopmiclt Th HCR <w aitaLh!int of inuhiple H(IR)rohe. ) a labeled.nokcuic. which. C:afl ttiiit. ff1 atipieation. Sroac.hastk hJ.eiing fbiovced by 1.cg trtav inetease 1.1)0 stsitivfty of (t?tC(t!O1i,juaks 311ü 0 uanufc a1on' of Ote Oh ki& eM id mok ole', Sodiastn allin,.' foikiwtd by HCR nty;eitase the a000rac2yøfdetcctidn, arwlyshç a.n.diot:4uantiflcation. of one (0)1Oi nt'eLie acki uioL'euks {00142J Ad uonal i,tcjhods arci app'wthl' f;,r agn& deLuon anU ptoinu of ni data are disclosed in. fc.r.cxamplè. US. Patents Nos, 5i4i$54. 5,54753: , 5,578,832.
5,/3'] ,7$4, 3.&00992, i$34158:, 5JE56.092. 5,90232* 5936324..5,98i,95.6,1125,601..
6 90 SY, 6 N 096 tsjs,o;o, h,'OLS9 R'1 0'L.md h2'S 615, 1 S Pa e'n Pob No 20040012676 nit 2000O50tC nil m Pt. I A,pheanoa PC I I,99 OeyOSP puh1uhcd as \)09 4 94) each of\hlch a so s hereby mcurnor ted Lw refe oce i n:nt,ret to' 41 [OUl43 Uctewon dnd'o quu}t Lalton f he labekil rnoe ale, ina Lonpnse cc' t'c of &ompnkr', in omp'ttcr c a (\mtpat so ft drL p odaLt' na' or s' a. o rLtdai)k thodhim hawiget puttr-txcc.utabk. histructiøhs tor;rf'.rmit tht Aic step$ of the method of the *i.nve0uon. Stuttthk computer reaclithie medium nc.i d.c floppy dLsk, CD MOM LYv U D\ ftROM h trddhL ci e, fla'h memory, MOM RAM me gacac tape etc Ihe co pt'tere.s:cutae1: mstu.ct}on\ nu.y be \k nun ma saaabc' ornpuiei knguage o cTh'flhi Muon of se\'41 iafl'Lei't5 H 15'( tO'nfmuaiOfl d h olo7y)etnuJs are cks aM in lot cxanpL", Sen aa and \4e4'vns et d hn. hh hon o C' noutai&noI Bo!oç IL lhRis IP\VS Puol,t1mg ( oripan' Hnsznn, 1 097 Sal1 bc Seat Ic', Kao I (1<d) C niu (.1k/ tJup',)j LnAJo1ec&/ar aioJotv. (E.lsevier, Amsterdam, 199%); Ra;s:hith and Bueh]er, BM/t?aiks Bthc:s: Apoucwon;nBu7io?a4i Schncè and Matkmd{CRC Press, London, 200(Y) ahd.
U e,cvc and B' am', Bwpitcrmav, . I Pa cui (:a ?or 4rait nt ( a ond vteoo Wiley & Soes Iau, 2Ya od. 001). See. also US 6170 1.08.
[0Q1441 c:ovtn prornt puduct i4 softwtin3 may be ed ftr a wrietv: ol 4A pn*e d s&n nwnge'nent of data ansIsss and n'sn L'rnk. it ope'auon See, I. S Patent \os 5 5'ft$'9 5 25 71o 5 73329 5,°" h64, 6 OoO,454, 6090555 6 1 5 561 6.188383. 6.223,121. 6,229,9)1 and 6,308,170., Conipaer methods related to enotyping 1; sceJk txampte. 1.1$ PatctitPub Its 200502501S1, 20Os0244*$3 20050108197, 20050079536 and 20050042k$$4 itiófl'; t*:jIt4* *Ii*4! j}é*én$4iøTh$ Ovet Swrkstu4h as the f ttrtet a itints PObNoS. 20c30097222, 200 2004W02$i&. 20040126*40, and 20040049S54 to "is w:ofthtlabS&u*Aectot any pSa*hetoot coo::s s::. jt** w k:ttomStm can rI'r compthet.corr urea,. aflw&c, :Øtr, tSVot ticsSni: (001 461 tiw Sk4,: ,,,tThe Sn$uiiisk)n 9 . atation d wdfrornthe detctton at ó$..ttffPtGfifr tbMennKlørkwtnzmnt ia someisçihc itSnrntiS a%oñthmcso also b tratsmIued to another 4evtn4/or trtgnent Transmtssion rjtbc datWtibrtnatton caa ornpSc the tranaferof4aSintbntIori from a:*s'r, source to neoS a The &s adSabttSnpt*imsw Safi n,.(g,,ainthe4ame *pft: i,S$; (4g4a4*thicssatès tpufltà, flth*nt$Mt4.swn s,ry.qntbk rstbki Sin vus msf*re &;S,,, gmo*ote $ig h44a:ito4 iSk [00141J.
overt pøbtt4opoItot pcStoak!poS eornnntS (on eMdS*m1esit suek chandcls &e iopct wires, optical flbrta trels annmunktkjn thannols, and stovase methL S isi MtSjiiUSMI Skis An %1±age4radiowawn*rnwave,or yj:s8si, LOCUSt, Ana'tó """ ss!=s':,'S:tss eotSS'snd tnnásSt. y ISu.4awottsnth oo4y a$iig,wavt'rms pSbapd s$iO:,, :*si" t4 J* nitt' flu' .$ssm4'hi*m 2$ alterna & " n thetVii as dh4 and paSbautàMTnt1⁄2sSn.
edits flats Ma (no Et4040a *:tt tOOtø) Thei liS; iof±jl44SM one or tawe ntu1tSM to Sgnact ateasc tte o%n MdIS thy xampk;.a v*i* oisa: : otd4"Mg A Snitvkwa anabtftta c*nSpjStoia pstnct&Stesre pmvlder at a health twt manager. n one embodiment the cquehsbn ts bat*i 4* the eYitw tt aM! infdà rogardl4a 4i*stdiég4øsk it &SLbnSi that in att Sbo4imS tb4$tviding & concb3sion to * pattent, a!,gahh care pt*vidot of a health c*i tflaobger :n6Lit&tra1suassbnoflidataqynt*c!rk.
RS1 AccorSgJy b Stystdrn j tOOiS* OncnpcSNiintetn isnod$Un_S sampics fix the preseuct at ahsemc' oft bWogwMiy active analyte to pttducc dat reptchn* the:au4 coIMhe*i44 d4a..:pz tgtheapaLft:datüä apatla*i*hea4*cure IG vt Ai catetnanaget ibr Mk ng:a hsiS6a*ea4ic rcWew, ots1iã Gfthe ttas*dic 4iMS*duijñôk. :11 4bitith cfuflthJjifpitidé4!j pathS ahátth:tarruvidcr ørahkascrinokzds3⁄4iaSoncft4áatwør. a t*s*
:.adU1 aiia:jiajto 4$ü1$o4:t:s::Iiiu, flQY$ a amsysS:.(br44gitàS1o$QCkauieeteto aftapp8nst$tt: use with the stannmg scmsbtg systcm 824 to, Sr nampls. pmducc a resuft. The computer :ë$oØtj%a bcuhdr*bód: 4:4j4 tit:*4;c(j snl2s The 4$1$$ !9 W4: :c?U8Q!, d*dflflQ3v$k$flPt :4vict t d:kLStnmuseth *n4opS4nmnitrtit.
eonntheatht CM be a *tOrdu*tk3 j:dn:zàrn.$ :jtjjoja r*i:à*s; T: an!d, eM Th4bA ** jjf frmUingam vcSing dMa. fltoun4n'm:cLm'ompbnnon4 **$. os Sksd ot1*h1 1 tt*tSU d6Ø* èitb A network connection. a ekss>nuectioww an inteme4 conntkat, $a &cnneaion can provide. Jot cenixnunicato.r ovcr the SYorid Viide Web. It ig civisianed that data can be tthnmitied tVer such iictwotks or ctiinectou brreception.andior tvIéW ty t pttity 22.* *Cht tëcci*vig irty 22 cnr ht. but flOt td k. a:pitietu, health irç :p.rovkier o.ti.
healt h care maiiage.r.
tOOl.53! Th. &ie embothinenL a cohputerr&hidabk medium inchidhsa medium uitahk thi *trans.m.isaior of a. result clan. analysis at an ei.iairOninCnta or bicitagca1 sarnpc. The. fitch urn Can htdude a resuli regarding a dase eonthtion or state tim. 5u&ueet wherein suc.ha tesalt s ck'ri. ed isinv the mthoJ c; hcd Lit ifl I I ( OTfl)LLtt I Is. aicibfs. rnedkx an ion-tttnisitdr.
19411 Ml Data A5tl w, In sonic e c'dmenk d'& scannei amu utnent pic>ducts c i intiy values fbi each p hon on the ur.uIy as well as ihe background intensity. Many inethed.,s can be used to caku.hite the nuMber of molecules in tbe sarupile. For examnle. the.
y4 Luta tbr t.hecoinrcd pos.iti.ons.on.the array ate.eho.ve.d, fto.n the daiset. and a, scatter pint.
gc;n1cd j oic t an in at the dais I his nev oct it vs ith I) vs itheut ih is.Lround nkn\It\ suturacted from ftc l4\ JCL4 A. Uuc',hold intevn. aluc ecu c swuHi'.hed in o dci to classify the positive spots and the negative spots. AtLof the positive spots are. snmnwdto pmvidca. total. caunt of imiqile stochastic labels. This process: cart be au.tcma.Eed in Microsoft excel oi' another cOmnuter software pthram oftlccj A i hLrncflns. to ihi uatc'd) i the uw of ChNlti HH' n'onihim s ft cl's 1⁄4fllL5lTh cicunu K-means chtstenng i i method ofckv5tet ank ci's vs inch aun to paitiflon al of I he tibsc i on', ii as c. ieser,i vhic eat. ohs. i on ht long's its the u'sc a h the nearest mean. The data can he pth into or 3 clusters (or marc, 3 elustem cni to product.: the.leanet ntunbcTs s&&riand the. iuniber of diia.p&ints ian be added. up to de.tinmthe the counts..
100156! \ I target molecules 1001571 J e method', Lu's and systcns WcJoed heic n a; he used in the &ha i,abeEin. of ino!ecu*es Such molecules include but are not limited to, polynucle.otides and polypeptides. 4 used herein, the terms p.oiyuuclcotide" and "nuolbic acid molecule" i'efers ic a polymeric form cf.ni:icleolidcs of any length, either ribc.n.udeoiicles, deovribo.nue1eot.ides koek.e.d nucleic acids U..NA) or pentide: nucleic anids (PN.As) ths *ccmfle.pwine and pydmidinc bses5, or othe? .i*tural, chencafly orbi.ochemicaily modthed nuraturdi., or dcrivat.ized nucleatide ban A polymieleotide" or Thuc.teie acid.
nolceule" ádi codsist of ä.singio A.uelecstide. cr hdsc.pä.ir; Ahctttd.ieiy. the "jOlynueldotidd" or nucl,eic acid molecule composes two or more nucleot.ides orhasepairs; For exa.mplet.he 4.'., polviwctuticc' u "flucLA co no!euUc ucuprics a L'Ll aboLu 2 3 + : 6 $ 9 i I I i2 13. 1 i5 I IS 19 20 4) 4O O () 70. () 9(1)Ø {)O UU 4Oi 00 (3Q 7iQ SOD. 9QQ. ct OOfl I1e,tid(tc or base phs. Jn aiOth.e éxiithk, t.h»= J)OViU-ik0t1dt cnpriscs.t Least about 1 500. tOO. 5OO, 3OO).. 4000, 4500, 5O.O. 5.OO. 6bOO. 6Of.
7O(O.. 7500, *OQO: 5OC 9000, 9500, or 10000 nucfrottdes or base p.afts Ti hcUiom 0.1 (he ts_, \vJt eOt1CL. can enInI'1e \uaaic and ioup. ila' tpiady l' ftnind ic RN \ or DNA. n mod flec' or sah-tnuted su,n u pI osp au' .nouns A. polyuu cindy t) jy comprscmocl IF1ed. imdeotides, such. m rr&Lhy.lat.c:d:iucIc.oti:des and nucieo.idc awHogs. The.
sequenc. of nutlcotidc: may be ifitrrupted hy tionnuc1eat:4.de. components. Thus. the tentis nucictdde, hucleotide. dexymi ciside ind ckoyh.uckot.i4e geneally iluciud.e araiowc stch as those described herein, These analogs: &ffQ those::UhOiecUieS havmg some siructura ftatu.res in. common with a naturally occnrrhw nucleosi.de or.nuci.e.odde auth that when incorporated wit. a. dde ad.or oligonucieosi.& seoen:ee they chow hvhichzatioi with a..ntitrq.hty ON. UTTU I' a a. d qunec n olunt n Ty pa. a fly, thc'\. a aog &e dt n ed from natuzJ> oca tIL nudcosidc' and nuelcondes tv tplac cad or modd\ m,z the bNe, the abase orthe phosphod.iestcr moiety. The changes can be tailor macto to stabilize or destkbthzo h b.nd ftirmttthm or enhante t.hL. :peoii citvofh.ihrichzatin.n. with a..
complementary nucleic hdd. sequ.et cc as ddsfrdd. in soS:iiistanc.e.s,. the molecule. hrd PNA, RN.A, or flNkRNA hybrids. The molecu ins can he inoleatrandc.d. or doubksfra.nd.ed:,. tin Iñgtances the. ni&etutes te RNA môinu.k.', iueh as niRNA., tRN\, tRN.A,icRNA iRN \, s RN A, m nuN A. 1' e RN A. r'olec uli can k pol uiei Litcd \ hi. rwt o h, the mRNA.mo.Ieeuks are notpoiyadenyiated. Abet ati.veiv. the mo.heulcs are DNA. moLecules.
The DNA rii.olecutoi can be. gehorhk: DNA. The DNA mo.lcculea. can. coinprisb ewris intron,s, untranslated reglo ts or any combination th.ereot {GoI5SI n aena. ntnc,s the mo1x L" rc,1olv14\Ides k u'cd ierun th tna "potypeptide" rr.fers. to a molecule conprising at least nne peptide. in som.t instances, the polypepti.de consists ala single. eeptl.de. .AiIt.c.ruatively, the.polye.ptid.e comprtses two or mere pepti es, For exainpte. the polyp.cptide domprises at least hbou.t 2, 3, 4. 5... 7; 8.. 9, 1.0, 11 12, 1 t, 14 1 I e. 11 IX I 20, i0, 40 MI bO 0, 80 90 100 {iQ tOO 4th MI0, boO 10, n. 90 in 1000 pcpn& Fxamnpes ol pnyc'pndc' incL lit hut.no nit I' tr'c'd o, ammo accls picans, pepudc. h'otnioiec,, 01 echadde', lipid' ii' eohpd, p*iosrhol nuls antbodics eniyn;c' ktncses \c.epL'n, trcmzupttc" litetoN and hgard\ flU 59 1 no methoth, ha' tnd s' sem i o,doed ha cm cac be m,ed to,toclv'ttca Is ltet individual occurreir es of identical or nearly identical. molecules ancttordifiktrent mckcules.
y1diitiahtta the th: i IatMèSotéMfttSI thfrø*Ø1k to 1* !AW*4:e4.W$M at k4A abouta abdãU0O9Q 807t 604 S04%* zS2&2$., Ifl 8 t 4,S;4S4oe1iue1àctj4o;:erbuepaI$. tniomoiusScs, *cntIca1iobe Iot e*arrh SnSWStSkd mq thó:1i$ i1w:*is:ao'..:::4d:*iMW;Lno1:* oS1& $à tbe1abottd cesctSantsihEsoflt hwacsttCatI*thtISt is *i'cbasdceUy labSd hi other hutancn, at lest 2,, S,, 4 5,6,7, *, 9 ør tO identicat or nesØy :idet*WLJQ Icuinat stoclu JIyiafrIed.AI@n41v4 at kSflL 3*c4&$*,X4 *.
01;i020 300; 400&:t%6OO tôO *ôo 9ooet ïóöö d&ions4i4sS ti, krnilS h:wIAb*tThaftt i.si.s.. at $a*t TtZ$ ; Z1Q(; k*.
3500, 4,00&4500t $a)0, 6,000; 7,0(X), $,®0 9,000; or 10040 itntwa] or nenty identical njolccuks are stochasdcaliy labeled k other iwanca at least I $,00O; *0004 2s,OU; I34,0G43$,O$;44.(fl;:4OØ; S 0006O0(ö *fJU0 000;9t0&t pr 100,000 kta or risti. i OhtiM fl!o SanS 0th tk2ItyIthtti fØOUOj:M:t:th, ccii*fldti4iffienL n*lu1r*uSobe1ódwniptJsø Lei& than 7%, ?0t4 $$% 60%. 5504 50%, 4$%, 40%, 35%, 30%, 2$%, 20%, 15%, tO%, $4%;,2% 1% SS:: kiy ThdiA 1t:niàM4tSy 4ifft4::ka:j4 1 ZL4446,:t S5 9. iO iO; óM, obc74* ós 14t1nO.itUckbks:Ot ba& pan. Ut sans instaces, at least one moleeule s stochasheafly ldbeted. In other instances, at kastZ 3,4,5,6, 7,ic torlUdnntnxtleculesares*cba*ttaflylab*d Altcniativdy, at toast 20, 30 40 50, 6& 70,80,90. 1 00k. 2* 00 400.500,6*700, SOOt flat, cQO, 2,000,250% 3O00, 3500; 4,000,4500, 5j)0& f,O00; 7,000, $,00O 000ot 10000 diffErent sk'uit at st1anfl$o&,IEi,otS I**iix*cE; ai'läast i500;2000O; 25,®fr.
3&'1'*OQOMt4*'U": 5G4Xt*tit4 *w oftbø; or naoiSes nosts',àá"L [G0,t6it: Tht,,*it 4' uW,t4'$ibeli": ,$3J.,, p':,',",.
tons *ra Gwttt For as pletheut atnutw*±untot' inletk1c: ii $so*s!i" :S'rnca, the once, ,fteaonmow,,4at lead 9flC uac,ile Mtheiiampkhat iea*tabnt ts4Z4$6; t;Oiiti, U*I4 i&&zL3/* 4,.iöa 1*,. 8;cöor tôbtOt U greatertn 4tncnatmn:9f áit Miii Mtit*sl Pit oth Sled lilt ajttessS:or a osohztba! ti kie k SançIt Thttn*n&attnor aznoSot :tf th Sk t:ibaS1boitL zac44447;L 9 iO fl:çt2 a 14M eotICeMtatSOt anunt sf 4 tatv4naftt MG ub 1* S krflC to. *61 iii* Si Sm,ttfEbóula 16 k$ .SQHc'thJ z'* n)ICVU1C to belabotSf van be jg sa$ ttwot4antplb% caoSn different amounts or *ottentiaU1rns of the molecsles tote lix sotxc mnstances the oouqentS on or amount of óm d4cuktopsa ScencenSnn & thFth Sti ale ia aSSnt saEt St j*MgJ* aaiM4 a moi& pteat* sib rnpLn can üdñidUEssS rno$ui inowinp1c4aube:atba. os.14 $;43&i,49, 10, U,* 13, i4A$ 2Ot:2.3O 4%4$s K60t! * * Out iOO&mottfrnetgn*rtrtthtbcohznnrwioft amounjotom: n*bcuiè konearnpb can be Sn4c s:s onrnostothe: 4aeJeSfl$fl:tel:lwattfrntatflt $gá ala: p $IL4u' i* 1t:b4ng *ip14 fl: *iiSp sa:1 Itatk about Z5,Zi4øS,6j14 LiA 10, 11, 12, 13, 14., 15, 20, 25, *35, 40,45,50,60,70, SO, 90,er 100 or nEiretunes less thin dztt'brcn% tonttafrationsor aitiowtts cia nx4ecuk in two or tuort different samples Li ted tOnS VxLOñgmutekct lags 1*64IM nø c* :IU$dts.:taS, v!='ds..: *W4W*is:i4:ai phirahty ofahgrnniclcodde tas The oLtnuckotMc taos can cotnprLsq a target peeific eson4fauniqw3 tffiinegioi. L:ad tetMgofl *uz:pnth:bsMgio:regis*a iy:oiwiti*lis*r Looi6lAahn'SFX. iOthg:ide.:S1S)maytpIsminrsai primer bwdwg sth(1 001). unique identx&r region (1O0 and a tasgct speciik region (100 Oftf*66j.As shown in.F[G. 1 lA. th oIicaon.icIIeoEidc tag.( . 107) can co.r.nprise. a ur&ive:rsal.
Øri.met binding. i:te (1:io.) a utipn idenrifiet rtgIon ( 1 1 O3 and ataçt spccifIc region (I 05) h.: ita er',a flflflC1 t3lflti1P \t? ( I 1O a phosp iinkac' a.. dcpicted L'y an *" in. FIG. I I A. ;\ .iiown itt JTR. IS; hr oii,ucknide tag (1 128 can u'n,,rne a uru ci s il p reer hmding te I 22 a uniqu bknotkr i:non ( 123, bnJg.
glint I I 2) aud a ta.get speduTte cgion t) 12t) A', hu,t in FK I K the oLo ue1eunde tag (1 flM iPpn.se ci. ufl\ er',aI pci ne' h rd nt stu I 1) imni ur dentr'er go' L1 I S.) htaton Cctlct (I 1) 1 Id. Lugel quA lilt c11LCll. (11 S') A', Jansi 1 F) th. a' gonnckotdc ag II P tav ceriçne a wus e a1 jvi iicr bi tdm:e (1 7 tlidqw: identifier regiOn (1172). ligation. siqute 1 1 73), and a DNA talget)etft. sequn.ce (11.711..
it 0.167! As ttwn in FIG. i2A, an o.Iigonuckotide tag (1201!) nat comprise a universal prinef hind.ifig site (1.202:, a tmiqu.e id.éntiñi.r regi:o QoflRriSflQ a degenerate sequence 3j and target speitt ieg n I 204') A' ,ho si, ir FIG l'ft an oiqonuch onde lag 210) may comprt%e a unieisa1 p ine ndu1g uc 12 a un'cuc tdcnfle eglo (1215) comprising a degenerate sequence (1213) flanked, by two flanking:sct1:uOCes (1212 and 12.14) nfl a targct.spccitic region (1216').
[OO.168j The oligonuelcatide. tatcmav he. contpnse one àr marc stthndarvsttuctares. A shownm FIG the o]:igonncieotkkt uneji 30.1) comprises a hairpin. structure. The oligoiiuekatide tag (I 300 can eoniptli a iatg.t s1ciflc P.gionXI3.02,. a deavalik (I 3034304). and a. unique id nftlier rcgkm (1305).
i0Oi69 The. oiigonucIcotide tag:may comprise a target specific region that cau hybridize.
in a phiratiiv ofdifferent tafeet mnolecdks. For tniantpk as skiosn in PLO. I 3B, the ohgoma let tide tag (1310) cnmprises.a n iversa) primer hindng site (1:11 i) unique identifier region (13 12y and a Uiu"LrnI arget p4cift rcutor. (1 U3) Ihe uir\NaI a{gLt pecthc region 111) ma> ceinpi e an ohgoi. I equence that enabi.rs ishi idi al on t0 Lsriet molecules comprising a.polyA or polylJ squence [001791 A m hod far SyntheiSirtag a pitirality of o(igonuckcotdie tags 15 depicted in PIG.
0. . As &:hown.. in FIG. 1 0, oh.gonu:ekatide tags (1004) ear he synthesized seaaratc.t. The.
oh 5ourckoudc t <s (100$) ciui compnse ci riwer'a1 pi irier hliiLIBl ate P0) a nan uc iditifitrre.ion (1002.). aid e. prget sp<c.ific regiOn:.. f Ic itidi:vidutI a! igon teotide thg can N. poeled ki nc1duc& a olin nit v ot oliganu s co u g (1 00 comprisinsz a pLir I v o eifNent umqac icLntilici regions {00171J A netnod tot \ynthe\izing t plurany at o gonuceordie t ai is dcp.ctea at I 10 J...
nA. A howntflb itAdgonuctSide frntOt}eanbyniSSscpavi4 O 16 t nprI*a:nnlsrenat i4flJø).'P$ :u$àI:fri$iOIt4pt: .kk* (1 1j) :tdtM$ ap&phdrS&tbttasfdeiIctd yi&thflG. ilk. Asthow ntc 1srni>t:nt1ss:nntE oflgonudeatide:frasmcSflia4): rbcpk alityohh*aiàotke bgments (L1U4can&t jiöffió; 14ji4 $e lig*ø4 tn1t:* Srgt::ffitsion (t iS); 9 and; eslàadM4ohe r4action to remove dnn4zgatedpmducts (1105, 1101) The ofiga4eot3de 14$ (1 1C*) QOflT:$$13thCUflWCfSaLffIflS binding sittO 102$, uoiq ddinEificr r$in:QiG3)S oFREL J1&d%e3*phosphategmup*oxnthe hgfled*%oDUt*Oddet*O106)cflbe pbophate smup can be removed enzymattcally, t,or exan'iplc, a U polynucleetit kinase qat be i 4 o removc: thet Øo*ato rnup eO1 AathrtSi titsynOasiing igmStü tas hiiktcd tfltL i1tBAs sbownttHO llRan&aauekottdctag(ILZS)cambesynthesizcdbyligatwgtwo t:tln!ed tf2.
**npMtntisi$Lprin* Wfl4tng sito41 tI; %nqut1denttherNgon:QI23)as%da iø tar4 ttakg cific:rcgt(4 j, k1gasc. g;1t4 bsA tgstyt nbc u tOcjih;tW* oligonucleotide fragments (1121 and 3127) to pwducc an oligrnnwleotide tag (1128). Double standS haUon oflhe St splbu fl124> and riØit splint (1125) can. pmduce an oligonuclectide tag (Ll2) with a ltd4e splint (112*).
100DM Uflthflw OhgantiIeotdc*4gmenth i8:depiótStPtt tiC AüowntF1:O;ilA an:oi%onSkMMC C' &igonuclanide ttagnat(1 1fl)maycoxnprsn univcrsalpnmerMndinne (1 tSfl.unque 01* * :*t*4 * óW$4Stifl*m*1:* ffie:!intø se4(iifthttst ogOtUclebHdrnC*tQ11O)4toffipbflentofttt so ns4 A (iiS6T*tgoSiSt4dft4jS 0 1) W$S conse*:3Yjihospbatewhkh:puents xtaLtgflhe oligouucttotl4 frIgxaeni. Al sSwn B cjçp 1 ot 11(3 J( the huun tqutnce' I nd I i5-4 ofthc to frag n ients may anaet1 and. p<Ivrne?ase. can be u.sd to. &tid the 3 end ef the first a.guieit (1. 1 SO) to trod,uci Ø.tiOflCiCQtdC tag (. t I 5.8).
&4wont dCOUdL L ( I 58) i,,n oc'mrr1> a mi% ei aI M icr t"uding te I I 5 unique identifier r&'dn. ( L52., ligation séquliie ( F!.531h find t&g spedflc scdunc (1.I57). The xtset pcdie t.t it.icc Ii) oithc,hgonudcnu -IL tatj (11) nt no the ompkinunt or the onipknietn of the mxci spccifk n' zion I 1 5Y the second ol iow en u'e 1rrrnenr C I 9) 1 Ii. oho ilL k'OiJLt. Ii an 1K flt Olfl't Pfl C lOt. 1,t)L' Ii 0) ttfl lx' Tcflio\ ul to ii I lit obuoni c kotide w (1l 5) Pot C\ rnpk the label I)fi rn< corn'i hioUn and ohont ekot'de hawnie'Th. U ° comp is rig the houn hel i) 156 an lit tomcned srcpftwdin capture.. In.another eampk the. label (1 156) niay conirehe aS' phosphau. and.
oluouuckvudc Iragnierit (11 9i LOhipt&sW4 the)' phosçha{e I 156) t.au be lelliosed Vid an eqnpcia (ee;, Lambda esonticleasé).
[0W74! A d pick4 in FTC 1111, fitsI nhgonnc iec'flde fi agnei I I 7Q) oinpi rin a u m.. eisa pnmet thudinr site 171 1 unique idenutiet tegiu 1 lI21 I lust &ieat lOll,X UetIcC (1173) is annealed to a seeondo1i:onuc.k.ot.OIetagnient (1176) compnsin@ a. second.hnauon sequence (1.1:74) and an RNA. eontplc-mentol the t&get quence (I 1:75) Sum 1 may c:omprith:nnSRng the first ahd s&ond ligation seq andes (1173 and. 1 F74.) foliowCd.Fy J c,t',1' tianu pnor of tO R'\A urnpk'nic.nt o I ii. qu n ci I F75) to pm 1w an ohomwleoude n (H 77 comp hing a urn e'a 1 r'°' bindtng site (NIl unique d.c'ntitr region 1 1724. a brat i&71ti00. ]41011Ce (1173f arid, a. target regio.n (.1 1. 75).
The oligonucleotidd.frag:rntits comprising the RNA compenientoIdi' target sequence can bd seiodfllvelv degráucd by RN.Asd. ththtCcnL.
!{HU 7S I ic ii nuw kind tat; can is at t.at about 1, 2. 4, c, 7 0, Q, . 12 13, H, u$ 16 1 15 t, 20 30 40,50,10 10 50 0, 100 200. 300, W, 00 600, 700, Sb0. 000 or 1000 r lcleotWes ot hJ>e pairs In anothet e\ampk the ougon ickotide ag comprises atleast about isOO..000; 25001 3,00th 35tiQ.4.O00; 4500. 5çQ 55QQ, :&00& 650& 7,000; 750th 8,000.; 8500. 9,000; 9.500. or Ith000 nuc.ieotides or bnspaira.
f0i 76! I'he tags can be hexam.crs, e g random. hexaners The taia. can.be randomly gr neiated fitnu a nt ntonom t. Icotidec The ia. i be 4senIh1rc by ru OIfll inl'poratng. monont;ikotide4, [00.1771 The: tags. can also be assciitb.kd without raridonincas, to generate a library f e itfn ent ags inch ai e no raniornk gene ated but a hch mdudc', >u ificient nuniba' of d.ifrent tags to practice the mcthods. 1.
It$wne odimcfltsasI Ok&OflUGiWtidC:iag can compäsc a cti*1M us, a aiit& S4utba&tM btt'"'ti,"'*th: àrnoEdor t"t1S t$ 1I*iMé :ói$ st1kàg& the'adoattMntbaák and £bt adde' Sgs cantontain :s.*,ft:'thnwMs:!.:','$' P'r444fttds' tg:i(4iáflf4he adtd tap'rnay',,ii r:ytzoüntM'tbernatr thargçt nab iecuks Mten detet a aflows s *rgSa n iotS4'S &S&caddd (OO17J The oil goxnwleoudt rag can compne a target spci'ffc ngiou. The target spct»=i& mJquk,isan mRNAfn iOSiaetsxcffii g n"inr4'n øUw4t.'ccvnice t1Sis bbnipkt"S atr d S A&r:saz,th"k tatgct *a!*.s:t'siM, v**ithnUé4*tcc.
tthc S tan S;n fldMcwtrTh'SuIld syn'ah oiacD}f' the xnRNA mokcuk+ AltsrnatSly, The target specilk rcgion tompdsea a sequence that h cQflY:.Pthfl, kttM tuttittifltectf Ic' region P*s * SMUtlC4:tl"iybtidizcd'*t I;l'g'"to iS iI4ett1th $$4AfW$Suifti ôa41 entbIGtftaChrnthtGftheOtIgonuClcOtldb t4"$i$It4$*bfl*'t :tthcsedo'thg:ururp'anyoticthSst1osd4t*eth 4.
d'St Iigath$). IbIS 1 $pe4flt t$ibnØit:fltqubdo tMV!Qed "** Sp*trKP'eflyMs; Tfr ti it, spi"a cotnpriacatleastdbout LZL4, 5 6 7, R,9, lik'll, 12, 1, 14, 15, 16, 17 t8 1920,* 40, 50,60, 70, 80.90, 100, ?00, 300,, 400, 500.600,700,800, 900, or 1000 mrclcotides or base paks. In another exan*ft, the target specdtc region comprtses at least about I 500,2*, 2SOO 30* 3500, 4O00 4500.5000, 5500,6000,6500.1000, 7500w 8(}00, 80O,. 9000,9500, & 1:00" ot:bS]ii4u*' Th.,'tbly, ,thi"S'' 4"ñthi" s',tó,", "aotiS4O it,i O tio i:2o&dkthk& hae'vaiit 100I8OI,M', nø 4tapy, t,: t"' i:4'ui *f $ gimeor gcaproduet *,ot5:_( g "pwdut."H thret,, ,, Stdct&gsansLy attach totS a*sle cmds g'Wóp ólfk i"và'L u*ta,j eaio arcs Øttgi4":4t" j".' ma kcu'k' comprising, a j1yA.sequea,. baShes ennsplt th'1&get páific:regioit :vent4es arandernseqxrnnce S eornplernexflry I aptürdity otdifftzrew mes QC:tU& Thpkoià:t*tn_to any ukk.*khaoq$ thfl Sü.U*#fl*"4$4MSj ft&Wgk t4ahSIthtüté óWi$ 4i*.*4fl1iS$ttSrStWS fOO1StfThtt4slipnuóLAvtde ra&dbsedisth'oMn*ontessSse1ti1be fl41$tttflW% :*S:If*S sptttby, mai thg catlnçt lstkMttifi& :rhi.can be nS&dIsthguè S*n M:0tT1 itilAt * &sTh tkhnUtyqWonIcoti4&tagtcancqrnp3*n ql1oe4n ó$4IflbS. seniicon4uS naMtt thtat coUtftSi Øpti 1$ Egen Ii *, antibo4tes sth II Stos, panlcks OK stazchat Mvuzg &ffbrcn( shapes. cohn batcods or ditacdon psftenis:MsbJfflttUhtItWAtt tStU*r*M.
pmtths or nutc acids, 4iffirett iwtopcs or any combinatbn tbenot The uniqut idcnti5er oncalrcoThpflf&G.a dcgenemUvc sa ThtuMque identitiez?Teflhacaft ccsnprI$e* ffl20i® 4G'øW7Q4 8%i VfrY... *4Ss*aO:ainf;frL: aóitnnarnp1eftsuthque I4ikl1e' rsflsn.con 4ts aiSt iboui i3*ZO; 3p00. 3*, 4S 4504, 5,OO0 ssOO, (r,OOO4 650O, 7.0* 15*O MG% KS 94009500, it 1:Q;X:.4uøjMorba$t W*4 P br,W 4$ ji4cfltfj i9Pamp1$t& at ten about th40 ii:4& :*2MO itu koddttt Ssc paWs
I
slit thttw$ver 1 pthnerM. ding MtdoniheattaohrnSota unMSpdar to the Sot Iabekd-atpØiton. tfth: Lpthnte wSt:zheartsS include, but arc not timited to, 47P (M13F), aJF3MF, AOXY, AOX5', BOHr, CMV_40, CMV -50.. CVMI LACrnt kingO gtI0F, hrntda gt IOR lambda t1 IF. iambdaatll R :M3rvZ$$I.2 1, i$R4%&1 i*k iO$EQ pQ p 42Q4pt 4 Eorwa4 pGL_kVpr3. pGLpr2_L pKLACI34 pQEJ& pQEJtS. p*wjil, pncji2 nssiseqjREflpe4 Ssujje &m $& *4&Jit $$P&13t1M, n::MmtsmJo aStofS;SvgS:p*tunttsa prim Siingóttanbc uaadfbt ampWkaa dotecthrn. wtdIor scqucndng ottkc Labelcd-mokcuk 3flWOr lab*d-ampltcoa fl:uiwaprinthtthngs4tnvwprat:Eeastabout 1,Z3.4,&6ts9* it. :is
t2i3t4. $, ikI.xt *: iA6*3,MQ So 6*: :OôiOt1G1tt$1iL *i iiothoro,ta Q1); Z eaJ$th,; 4A%4$øQO*.
5500, &00U4 65O0 1410, 75O& tacO; ssoo,. 9.Ott0 *5*iw 10,000 nucteotadn at base p*n.:?th174 w.W*ai d* $$diaEpSP:' W-SQ *(t4* ày (001 03j The øligpmzcleothfe tag may conipnsc aa adap4zt vega The adapter fegkrn may aabie:bM1sfl&ocbStt*pflbs The adap tcio*say *sbkh4MStsof (00184J: 1*01851 Tht Clu*MUckotidc tag n*y act as an initiator lbr a brhlttton cht tscdon 8C)tcrngifThcligonuc1vodôetagmaya4wiaiSzJir HCLIb tiini Øtin*indhigittrny abttk iziMMor f&ntt ussr: t:MtttitW I duMcSsnde4, thSIgo:rn*MSefl1Mear Mternativdy the oligonuclcot* tag comprbct a secondary structua ib used herS; àecondáiy.stAvcSh4es:tórthqySevnaxy, eta; .jttSget In;aomeinsia,ce, die °°Y rnruetwtis ahk$ a aein4ip strscture4au *t1oqpi bPtht *t* ui*: ut..;$:9ti Sb *tS$vâdëift* i*k*e$ * t'Ebj1 l$PMc*n *:: *g fr: S 1: aSSS1i.r$ I 1 tuO$iS. Is: :ovàSgo tot a setIeit.i isco a to The rue kt4e to wbkhS oligonucteoude ta is atiached and the overhang sequence hybridizes to the ralecute. N> :9ysguntai1tjS: thtniSbS aóSas attrnpiateb &p&ymerase or &.esbsdSts tl* tag: tornprLnt auàie&iorsyutMtonsietisdsandionmthflednuc1e1aci4ek4 IooiRtt:SObgonteotidc:tn1pthingaSirpiiL mayacias arobe*rwhySzathn cbS': F hun aothasbctabcL*ndhybnduatoncharn is w(i3Ct)nfltnd comprising óctiSica1t 1abetingonc:ornre:nucIè:aøi4 niotecules wfth an:. jft jtidttj wi*eM i mutSS:tagts:hah$nan4 tk 4t*ó :I PdIfl* ShcSkaTh Aniâ 1 bdhtidSàn:rtad&nis dqcS: in fiG: MMihwa : RG34$to U WE: ZO 4 ØS::th$ øSi. witit t: pturalI*y of hairpin oizgonuctentide tags (34901by iniklatln4j a h5tSwatkm cbazn reacPon.
TMSpiaoiipnacSddetags may:eo&npilse one or more ti 3% an evethang :(M2O4*')it:skØt4àaflto 1i:!ucI*k14: * Meat tu *M& TkIm4i: **Ja:, :44:'& fr pi*4 hia$U{thdtb* n óKá jio* to skthIc teb*bnS4;ThnpItttrnpsrtt*4nth or mW 1caS:ntet*4es.nias be acid molecule hybrId The towrbat rtspon (3421)) and the ncm (343) may b cem$cn)entary to the one at more adaptea.. The loop (3450) otttw dligonneleotide tag may ollpiwdeotk(e saw The Ifrsit hneathcd ohonacIaflidt tag orlabe1d nmiccuiA (3415) thk act at, a!üiator tbr hybrization eta second hairpin oOgomckotide ta to tI* bbcled rnol$uiè (a4is; to pducebekdmokoik with rvo linu izetol teotkk tap (34$S*secottd lnszted: o1ig.üu leoti&: t:tafl aatann intlit*b;atotS hybridization reactIon. This proces& an be repeated ntltapk times to pm duct a lube led ttAeCtdi: with mtdtpl:iSathS IGCkpScspAiATM S(4ffi the: Ifl pmt* an enable detection oftt* labeled molstule The tabè (34 IO M7O) may be any type d.(g,:flwq*s* I*4t ($41 s4gw:s4i:t tollonuckoti tf$414 S 7gIig:iØØdd *hfltithàhC1iL4I(34iO,I4iO>Sy bctthse one ug$ piS Sip i,nS&a 4Seàihk*t*itor flpie,i bipiw *:*si 335tD:St tipSsai*c:. E:PIa a14SD4iQ 4Ths ASnauScthatSayt.nparIcb. e?suLd it: bet-based can;&ab1 alabek tsSuie wSóbean esuk M
I_ -
:tteI: &tdSck sM:*t4* q$USdt&$tik(iS Oftf88j* lnsomc coLnrries:a C?cSt &XMJt 2, 3, 4, _5, U,, , * t) Q, L'. 20:s, o, 3 o () 60. t, ?fI, f5 80 O. ) or oo dit 1 rit oiigOnnckotide. L.ui,s. IE otht i plirlity L"fl)I 7L 4t kat nbcs 204) OO 4QØ 500 600 700, 8O OO. 1 (iclO 000, 000 t 300 5c00, 6 bOo 7MOO. 000 °,000, or 0000 W&cnt gonuWotW tas ttin<rw:Iy the rktal t ot uhorn dcolnh. ta cmpns\ tt kas ah&ut 21' 000 0 000 40 001 0 000 t0 000 70 flOO 10,0O0. 90000 or 100 001) difierent o thUS9J 1' t' no'; b i o olironuchntidt tas in thc piurahty ol uftoin;ck'n UL L,s is in e>wtss of the nttrnberofmokcuies to be ktbei:ed. In some thstuiees the. eumbet of ohont ekotide t tos m { he p utiJuv ui & uno. Lunde tatts h fl ow 2, 4 5 t 9, 10, 15. 200.40,5.0. 60, 70,. 80,. 90, or 100 times greater than the number of molectdes to be labeled.
0.0l90 T*. tiquther of diffèren.t o gcuuckdGde tags fr.th. plu.raihy of oit.gonve.iecithiè.
lags ii oflea..n excess of the number otchbArent molecules to be htheied. Ilu some instances, the number ofdifirent ol,onuckotde tags in the plur&ity of oligonwrieotide tags is at least about, U, 2.2.5,3. 3.5 t*4.5. 5, h8. 9, 10. iS, 20,30.. 40. 50, 60. 70, SJ, 90. or 100.
times ercatci; th n..the ni:rnber of thtterrnt mck.e41.ics to be. la.bekd.
[001911 hi semé iu.stanéeS stáchustk labeling of a niokeuk óomptisos it plurJ.t.\ of ohc:ontcittmde tags.,wheretn the cancentfltiou. of the diftitrent oiigonuclettide tans, in he plurality of ollgQn!adtotide ta>s is the. samC. it suh hstitflces, thepiiraiity ci oligolu. lent KU t> g' orrp s S qual d' s 01 caL h di lien) it (}h,OiiUCL i' u4e tc g AdditionaHy the relative ratio of the diftirent oligonucicotide tags in the plurality of ohgouucl.cottde I 1 00i92f Ii. some in.atanees,:stoehasuc kha0nt of a moleete. comprises fi)hiflth{y of ohonucEeoUde L gs. herun the concLuttattor of the ddfucnt c goaudeottd thc phu 4l't' ofotisont etcotide tags s J arent hi suei a st inc es the plurality of oligonuckotide tags comprises thfftrent numbers of each dffl:ercnt oligonuetcotide tag.
A.ddi.tiomW.v the relative ratio of the <.ifcrent cWgonueieotide tags in. the plurality of ohgonue.Iemid.e is not 1:1:1. I. hi SoOt) instances; some oli.rto.nucleoLide tans arc present at hhzber concentrations then other Oi.lgtntd.eoflde tags in the plurality if oh{$OUtie.StKk tags, in sore.int4ncei,. stochastic labcthiØwith difietcltt eo:ncentrttdons of ohtqlmefot:t4Q tits extends the. atmiple. measurement dynamic range: without increasing the number of dithiient a icls used Ioi e\ample, con.sicei tothasneaU Ia icing 3 uc CtC 4c10 n flT.Z &LCJk' s ith 0 thffc. zu' ohLonuceor&c aU at equa concenti utlo 1 0 C"PCCI to \sCt\ c 3 a,.
A1d*$pc%tb6b* aLtO 1áL $11 i*eshl M* 44 jtët"S j:t: ót$bt* !à J:;:$.4Q ftiii. ;swas iitoc,wt j: * ts: tda;t&s:;ofln1s**ntta tbaiintnbcc ot1nkae.iakit, töSiteWh4rattWtSdestdboLeot1di tgsstejk&ye jléiti etei sø a w:* it, t&t'tZ, 4,: i, :g, thJ$* 25,30. 45,50, 55,40, 65 lOs 7$, *r15t 94 $, Ot 100. A1tCTMtW4I)f. tttti'tS totp tASCJZ, wbqr1⁄4 B, C.4"As4tàaSt,abS t Z41 6,t& tb. U4 sO 5" 4*15* k3aotitso [%$4f: in " s, *t,drtw1*;:st4i MSioñtkk *, wthe pkn$ty folkpmdeot4e ta$s 13 the same For $*P difIetcnt o1tgønudéotide tas the eenccntntiou of at teat 2, i, 4, , n durctcnt $4onucIaft34c tap is tie same, AftcrnatMty, the a rn',, flfioaØ,Snw, ; 4itWjonacksidoags ht" piw of 4OtThX1ttii$ iZ dill beeht. For "C difftront óUn$*tide:tMgs the *os tnttotot at lowt 2,344, ii diffirenz oligonuclootide tags t< diffirent In some m4tances, tr W5 41 ntougwnnacotid':ap, thø 4n'1waotueMrathn:tbti làa:Z:4raiisZt otigontzeleotIcle rags is s least about 0.1,02,03,, 04. (k5 0401, 0.8, 0S 1, 1 is 1 s 1 75, t 2z': Z.%:z1&3:,4, s. $k7U rn; is. zn':s. io, so,: otzOO.ic, 400.
s0Q;6:004io0800'%O,AtlflG4ak 4%, 565,1%, 8%, 9% 1%, 20%, zs%,, 30%, 3%% 40%,. 4$%. 0% S5% 60%., 63%. 70%. 7% RO%. 85%, 90% 95%, 97%. or 100% of the difftrentthgonutStt tag in the plurality ofoligunucleotide t*hAi:ThiitSótn'StI'tL AhStiviy, öt $%.4Y7% 2%r9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, S)%ie&S%. 70%, 5% 80VW%$4 95 W%* diftntOi4gtootidti1s'lulit'bf óligr.mudlcofit a85 laye a:different oncent*Ma tSoqües otS oKgonut,ietS tg*inaye mS:a:rnSmi'e ifl$,FF:,.4$4tt1!9$ t ta'SSofbiIgt',leoi4i M.Sh j';p; 1' wh*ojoaAit1aSe:": .t:pfia "ThtóJIo"k*td(ta coniprudng a universal pruner bxndin site (1501)4 a first unique idemifier region (1302) and a. IS th speMficreg1en(1UO3an2nncat to *soceud oligonxictetktu tag (1 magniMnEi rrbssfle G*S nnittr:(iO4 fl$5) Téóä)j$:g$fl4pj :21 th jjjjj 04 tjc *tpoion a.S+4*iisk wit Ike concSSitioFthtligenuSi4&t*ssethtoá*thatht:nunaI*rotDWA at$ S:oligoueIeHdnag &ri enA twtIflbSdDNZ ikuk: ijjf:.:.::t: ti:0fl14**A. 1:sita:.4o#sa.
o*ntaoiianqoitnoth lead tflhil:t:poiitvt& ikus,, the oiLo icSnag an be optimized to rninüSe ohigDóut6ot1dt t*g dither fixmatknL AfternatMly,, oligottuc1eot4e gs:tw4hna4iicardód. tà*dn*titida:ditnfln.
tbibi, Alesal s*pittet inFIG. I olIgonuOlutti$g dither to tlIttttód:frted*cób hiq*3j r$jft$*4P $1 I*!*4jflb*tkó :1* taj4 sequcne. M shown lit FIG. J6 un otlsenucleotkh tag (j6I *xt*prisin a primer bhgling site (1611% unique idcxtra rc*iot (L612\ and target eciflc son (1613) cmpr'$ aflnndaY,phpsptIatcteqpt SeSe 14tm s an bgieasedtd:MgnL. fl tLttAth k.
:sompá onea more tLSrwanfpthsen (i11S and i1*3.an4on4or.rnnrcieerse primea (1414 SJIth* spfk Th Vbc:i$S 4 ($1fl fl4L saai*g piIt(16i)spith&unsdfià rgk(it$o1ftigIStS ts*t:p4.: (1:614114 I6i$J:Th&t tan bt amplified tsüig a NIt DNA po1ymrases which at amplify ten$ate comprising one or more uraøils TIws any dtmerized oUgonutlstzdc tags tannot b amplified by NIt t4ñ981 Vxfl.Sted*welà& 1001991 Tht:dt';kki:S 4w*4$$ts4 ñóa4 S can be st to gcabley:anthefen any *hifremi:Nmc.* o'a:t à; ntPl; kic:.;*4$*k t*hth po*i:t:bci$n_ *! fl: Utdbd ttátiern'htm *21,tqUCnCtw$' ft rudiOaSiwk4 iiS pyrene:nrikiy. gókjtw wmkbSi tht:Sliistb4g itsstz$SSfSS S o o tOOObi, Tn sonth iastthcts4lnttMt &s&osedberM fiMh S ttactthgone or : *w:l:Se azuplicon). fle:rnetbuds:can. ftachh!fl*oornR%rc ttØabI4 k&±pt iabckt- ow41 fr 14 tIII*flSfr k:flo k Thi.i!=k1:tfr 4t.ø.fr:FW4kCr [abeiTbe tjt ljj:jc: liból CTP*tht*, Or Mo flu, in some embodtments the detectable Label hçftttachtd to a psbe which bw4 d* mokquka1thchsIqaja, ThSnocpw,, braamkaftqr ftcrajiorbbèd h bsn *i tdtt lit t t*iokcSWtfflto $j1jflgjg :aais& ntben. Thi:procen tanTing a Stb:x$4ilcpr*bad: MWk S:dcces th likelihood tbar a signaL i tht result otnonspxdlc bMding of a tabet or nonspecific bindiig of Snzolecukth the nc teCaO: :wMansad aectotp&sib*uSsth'ttntàic ahs IEfthib @fra:*eok4:1i:4$4o 4pt::ss*a t :tábebaimple, a n*olik sc4pnbe tbáesi bind 4 a k (00*1 ssas&siir4a*a&sdao v®:a*c?t 4rc; f1uortSdyto ü-CbR itIS. :rt sont S MS 0th (6FAMTh, 6xyfiuoLsceüz), MAX (MIS Ester), flETS6&TEX 6U.TYE66$, Th deicetible Sel:canbean fiuor4ye;an$s*tAiemHuSP dyes include Alan Fluort 48 (P4135 EMz4 Alexa Fluorl!) 532 (MiS £stcO. Alan flu,tt 546 4MHS Er) #a*flU 5tk4I4US;Esit),Aku flS*64tOEaiAPtàocit Mo 4i4tsitotMxa P S IS). MSy,thSSethttlibeUsa c?t4:4yj*. Exuñpleiofc4yS ipG* 4à: aat S *q cg3 C3Bi'$;$4 cØ QI, 1 ts,øt d*c iabelktfiootaS dy& Nón Urn tplsJtons (AS 4:Awgb.
Pb*SdT3QS(RHS13t)TETPfw id *ts*" kS hi M Etflà; LI'COiUkDg*tMtá *st fl' o'tDy. k,:*r 1itbv*CW (NBS Estet) Lu søme instances, the dttcot*bte label is rtM 563 Aft4nmrnweLy, the t*ectable jabel is C& Oft2:O3j The. detoctabk label. can b Rhodamnric d've.. Examples oirhodarthie dyes inctude.
but art not Jimitedto, Rhodanin Ete), fAMRA, iAJMRA1 " (NUS te) }thodamlne Rs:-){.NH.s::Est). RO)I1 (NHS. Etañ. t *i T*AMRAJ*(Ai. ide). th othe im, ia. acsk. thc d:ctcctable IkhcI L a \Vc.flk) bye. WIRU[) b.y. s inchid. hu &c: not limikedto. EM dyt. WeURID 1)3 c1e, and WeftREd) D2 tyi.
the IJect tile L ie IN L\a' Red -X \HS F',t) erJ' 14O \H' Ftu, oi D 7O (1Nn5 Eshn), t!}024] Lu some instances. deleciai>i kibek aiciude.a linker 2.:io1ec:ule. .Examriles of linker moeeules nelude bui uc not htnted to b.onr audin, t:cp d% dw HRP, pt&ten A, pureui C. at boles e Itgrnl thei titl p&yhtsndffle, Ni, I AU 4ag' mc g' Aliernativeb', dctcctabk labeis. ineiude.hea.yv metaAs, electron.d.o.nors/acceptons acridiniu.rn esters, dyes andcakyIm.etric substtates. mother instances, detectabe Mit's include enzymes such as alkaline phosphathse, peroxidaseand. hthiferase.
[t10205J.A. change in mass eanhe. consMerrd a detectahiedabel. ssisibe case nt'surfhce: pk'avon tcsouaace icteenen It. lled tb4fl OtuL. cadU eOlnU2C L5LUl JCttc'3b labels tha.t are not mentioned herein, which may be.empkwed. in the operation ofthe present invention., [002061 Lu semé in.standeS d;tec.thbld labels are used. with primers.. Fot bxinipie., the.
ulmversal primer. is.a labeled with the deteet.abkt label. (e.g., C$..lak.:ed unjversa.i. primer tluorQphoni:laoele.d. n iversal primer). Aiteme.tively. the tarsuet pec.thc erimer h labekd with the dk ciable I bel (e; , I c6 4abcLc agct N) Iii oLr r'{ai s S. detectable labek..aru used. with theoligonucleotide tags., For cxa.melc, the oligonucleoticle. tag is labeled with a detthd.a.bie ldbbl. (e.g. hiotinlaheled. oligo.n.ucidotide t:e4, in. otber instances, delectable iahds are irec1. w.lth the. nucleIc, acid tentplaremolecu.le. Deectable labek can. be u<>cJ to dehit the labeke-noleer ks or bJed'ampoiw Atematne deteLtable abs are used to detect the. nucleic acid template. molecule.
[002071 In some instances, the detcetaile ahel. isattaehed to theprimcr ligonucleotide . labe.lethbioiecul:c. labele4-amplieon., ptobe 11CR probe. and/or non4ibeled. molecule.
Methods br attachingthe detectable Mid to the pri.mec ohgonucleotide tag. iabelecL tno.kcttIe kbele&arnpl:icon.: and;or nun-Iabe.kd molecule intl.ude but are not limited to, chetieal l.a beflftg tt n.d ena met la.helitig. 3* j* the detecntbk taD<I is at.t.ahed by:chemnwai]abelingc In sotn.e embothments, chemical. labeling techniques comprise a chemically reactive group NOm-limiting exainpics. o:frCa:ctie grods inc'lddie adi.n.c:cabtivc suintnds) esters sued as \HS fluoresLun ar Ni-IS ibodamme amine aiJue 4.2 i'/J\4ht\ gictidii FITt and,uIfls&h 1iUiv.
flu.or s*uc]: afiu.oreceir.:5makimide. Insoni e:ibodiiient, reattlea bfat.yof these H: it'ti dy v LII duCL.. nn1rcuIe rL1N II a 1 h1e cin ittt 1ond torrii htic n a flu,rophor. *a rAd.th& iiker andVor a:geflL hi S()i11 tmho:d.imc.nts. die.retct.ive ioup s isotb cyanates. In soiié nbodimerits, a label is htfaj/Thed lb thi aert t ugh the priinury dtnlfl4A k,we idc ehain' k.MflhudIrflL at ehumea laic hact ft a \ HSLstL, thet..isrv method.
Mternadv&y t!ic dateciab]e label is attached v zyrnaU/. .iabe.ini.. .Enzvtnat.ie ntietimz ircthoa' an wcli be, Fat not hninee to 1mM n &.ero1 p naebtot n iLase \[sd3 1.\ cv c...rnei poteir p iophep.v tedien L UAI'dcla1⁄2e (t\( P PP Fac1. runan Li-L4iih t nfcae,n AG I) Q-tag tr>ng1u{anmiic (TGas ukleliyde tagiforuy.Igiycm.cgen.erating cn±yme, Pmtated pfokarvotic deh abgenase (llaIoTagi'MI1, and fitsy1dtion. iof/p dbih fihrnbs 4trahstèrasc (l>Efasc) methods. Affinity Iahet.ths cii ineligle. bl4t LS TUJ Inn.;ted tQ naneuva.lant methods utihz joe dihvdjofb f.redu.cusc (DHFR.) and Pbe3b\ 4 in itant oil KOo bmdmg nmem 2 di'JJPI 2(1 ld\ ) and met k?hchuion jiethods.
1100209] Cii Iin.ki.ni. . attats can he used toattach a detectable ktbei. to the t)flTflt, oh.gonuckotidd &., idhdIed4iolecuk. kub&ed-dthphdhn. and/hr on beicd.thoteeuk. itt SOT1W ih1⁄2,LiiC ic n,I"ih P heat aklehs CC Glut n ULch'yde CJfl h <n itmine groups to etcete trosskhks by $evetxd Fot eeampk*, under reducing conditions, the.aldchydeson: toth ends of.ciutanddehyde coupht with nniiues to form secondary antIn.e In ka:ges, 1002101 in. some histdnces attachmeiit of the ddtecidhle lube! to the irnder OIin;Ofltt%,Ietnde tap. hibelad-mo ecuLe, U1Ij and/or non-lithe-hid molecule eotnpuc' pcrlod4u>au\on 101 oned b icductt <rnmron hi onic mtinccs Suifo-SMCC or other heterobitImetional cmssh.nicers are used to conjugate the detectable it. thefl primer. ollgo.nucicotidc tag IabeiedmoIee&e talc e.d-.amphcon:. afl:d/OrnOftiabeied molecule. Porexanqile Su.JfoSMC(.is tsedio cçnig e an euzvthe to:5 drug. In some embo.dnnents the en me is activated and. purified in one step and then con ugated to Ute drugi:n *I{ .Stictstid step. Ilti anne. eti bob irneuts., thefl directkm.aUty ofceosstinkbtg: is. Ium.ted one speoifico.tientadol enii.fle$. on the enzvrño to std.fhythyl groups on the:. atutihody)..
100211] X. Suppfl {1102 I In om in-ice', thc unethock ku, and sv'tcns dtsco,cd hoiest cornrt'e ct sulweut I be teun upport and snbstrate a used hereto ce used mterchane&hh and refet Eo a watenor group ft i: 1aten.as having a.ri.d or er.nifigid.guitcc or s.uriwe*s.. ilic aippt>*n ot ub.stiate taa t solid.upp:hrt AItenttive1y, the:SUbrt S 1 tOfl4OJid rh %1I),Lftt (n}flf "a Tt fltt ntMane, fap' pbstic, CuitCd hce fl surfa:ce L4ass; s1id chip. or th1yCOiIbflatkfl trFeot.l many c.1Dbodimins at kastone "Ui: t obd \\ dl hc ukt in1 I. dthouLh in,arne cmbocluncrns t a be tinabL to rhyI') cp4te v th4>)ts cegksns O. 1iUCLfl corpond s it, fur e> a ph' " r sed teor,, p is t't. bed n toe cs or the like \.s. cotd"nz H other u butjrnenk the solid uppmt(' sill t hc tli hum ol beads testris, Is 1nN.o'pht'r s o±ei gcornett u tontigth 1tons Aherwtm eh1 the solid upport(s) comp es nhca Cups, mIuup.t eL, nano)aIuUeS, pJ ue'> and arrays Kktheds and tech iquc' ap'1icabk' to polymer ic.krding proteiri} array sy t esis have been. described in U.S. Patent Pub. No. OC50tj747Y SO 00 585lt U S i>ntwit No'> 5,14,SSt, 5242,°fl, S 252 743 32 o33 5.38424L $,40517Sa, 5.424486, SM 1,683., 5442,861 5.491,0.74,.5,5fl6Ri..5,50,2i5..
7I t<39, ¶7s Y S;9. S,fOS. 5 $4,71 I 5,Mt,?4. S,79S 71o,Xil,070, 5837832 5 854 [0. 5 858,t5$ 5 916 324 5 96h 0, S >74 1m1, 5,QI lhS S 6.025,601. 6.033,860. 6,040,1.93, 64)90,555. 6.136,249, &269846 and 6A2&752. in Publication.No. WO 99/36760 and. .\U C:L/S&S.., wi eb are. aU incoquint:ed herein by rcsferdnbc. in. thbk dntfrety thi' nEll purpascs. Patents th:at. dc.4cribc. s.ynthdis technklues. id specd7c entbodirnent.s include U.S. Patent \s. 5,412,05?. 6,i:472oS, 4'l6 6i 1.0.189.
5859,. 165. aiW 5,959,09S Nuci&c acid arraa are. ckcrihe4 in iiany of th abov. patents ii A tv n o the sana' ft cha ques i m lic applied to poh pptue at a Add 0 iunal cv. mupia y substrates are disclosed in ij:S:. Patent No, 5 744305 and. US Patent Nb. No.s, 20090 49340 and 20080038559.
IAW2.1 Jr. some distances. the solid suppcn.s a bead. Exninpks ofbe.ads:inckde, hut are not hmu.d to sttcptas idm hea. s, agaio'e beds nagnctL he KR Dn beadso& MAC St rniuobeads a.ntthoe conjuitt4 beaL {e g ann rnnuuuog ohu tn i. rohaaJ. pwtm \ LonpIg$ed heacs, protern U conug ited hads protem AC> contubated Nad% "otcn I. conjugated beads, oligo-dT conjugated bead silica beads, siUcal4ze bcads.nrti4'ilotth mIcroheaci, ant 4iuomoclrrome miembead, and E3cMaaM CnrhoxvThmrinated Magnetic iOQ214FTFc. solid supportc.an b.c. an array t'r micrarny. The solid support can coniprise ceL egrors The so lid support can be an addro's ible u, iv In son rtanscs, the ao a'> cothrises a plurality of probes fixed onto a sblid uthcc ih&phd'aht' of pmbc. enablks hybridization of the idbciedu'tolecuk and/ori.abeIed*arnp1icon to the.sohd surface. The p.krah.ty frobes compn.es a sequence. tha.i is co.x lpiefne.1taIry to at cae t a QfthC beed.-n<iecuk and./ot abeed*apIkon. i scrëe mnstant.eS:; the plur&hy ofjtcbes COtnIrise 2 that s to the Oi:OfltcieOtk1e:pitétthe:*beii&i-nkcu1c: andicr 1abe1edanipiecni. n other inst.aues. ti!=ura.i.y otpobea cc>.nipnse a seqwb.nce hat icônIemeta ry f.c> th uciioitformed oh2o*aue.Iecnid.e tag to the. iioLeei.Ie.
j 92 1 5J T1 ifl<; c lfl ( OflqN OlC' or flit1 t' pt\*bc;s The prob' can lv t a (SI totnictt, s cc m flC 18 \ ,hnsk ii in it 3 1< v-1 M( 1KG and ML tht ui r (1801 N06 1811, 182 l83 eancompriNe a rohc k3 1809, IU 1830 IKiel compu',uie a st-tjtLMCc tha s complenientaty to at kat a v"° of the uget km)kcuk (I M02, 807, 8) I 829 i83; aid qu:nce tLt i, eoninbincmtar to then iquc ick Th1ie ftg flil Ot O ohaeuuchxiudc L4! ti 80, 1808 1$ 1. 1 0, 1834) \ hon n HG tSA 18Th 1KG and 811.
the sequence that is co.np.emee.tarv t 1e.St Ierto.ri cfthe tarfet molecule (1.802. 1807.
sN, I 83 ar beaU lo Oic' an a A hon n i FRI I %C, I r u cc that m cempbcrncnrq to the ume ic identifici s.cgion V 8 12 Cci he attac cJ to the rray As 18D4$FJhe array(18i6, 1820, 1824) can compose aflrst probc(18i7. 1821,1825) cetriroan.u. cvpie.nCe that U cOmierflentaty to at least a portion of the *aqet: tnnieeuie and a eçpnd. probd ( 181. 9 1:823, 1.827) that 1. domemctary to the tthiqtic.idcntioci reio.n. RIG..
LKA$ 811 alsii dopict:thc va4nus.ways at which a stochasticaily labeled. molecule (18.05..
1.810, 1.815. l I 1822. 1 2& .83 I.; 37 cth hybridize tathe arrays. Fat. example, a 1-16 8 A nid ( the iura { ion at {h an ncu.. hkpufle?-ix a id UK a 1K t molecule o.ithc;stoeh.a:st.icatI labeled mokeuk (1 80.5. 18.15) can hybridize to the pmbe.
(1804, 1814) onthe. array As thown in FiG 1813. L8LRi$.FL there danbe a gap in the regiOns of the st.othastieadv abe1ed.rnoiecwe (18 ilL 1)11.8. iPr72 I 826,18.31. 1827) thatean k4'nchze to the proh or ffi s shown ni FiG ND)F &id Nil, thffcicnt ruioa of thestochasticaily labeted molecule (1818. 1822. 826, 1837) can hybridize to. two Or more.
ptobc, ot the flak the anay probc can be ri many thReteet toemat I he arias probes can comprise a sequence that i.s compiementhn to a unique hientifler region a St24UCUCC that: is ccut.pietnen.taryio the target. molecule, or a c IthIflation. thereoL ilvlti:di.aatinn of the stochasiica liv labeled nnjiecul C to ib airav can occut by a: cartel. of WayS. For examI& tw:ar more teeleotide of the slochasticaUy hthelcd nioleuie can hybridize to. one or more: probes on. the array, The two or. more neeleotides ofthe tocbasticafls labeled molecule that hybndu; to the probes 1fl43 be consccutn-e rue eo ides iwo-consecutive nucleotides, or a combinationthereof The stochasticafly labeled molecule Ilidi \ L1%d.NIdVCJ (0 tlLJ [fl()V tJ hL dctcctod 1S TIItAhoU 1'dk}V1'1 ft lt i I fU tX4 I th sUchisica11y kbt d makcu1ec can be diret.&{tCte& Dire.ttv thteetina the t.ocha.stieitIN; IEa.bkd xt' t.: may eni1ide d.eectii:n *f a Iiatefi ot dtectahie a& L. the stoch.asticat1y:ábe1ed moelc.uks can be indirectly detected. Indirect detecUon of the gweha&icaUy iabdkd rñd.ecule hty cothprise*I1i.atkhi or otit enzymatic or nen-euizy.matic methods.
j002 16J T1 iinv c lfl Il c eR ot hnnil ro e' nrpk { e a a an be ni i 1 $ J2-,$8-. 64,Wk 96-,.i2-, 128-. 144.. 1.50-. 11&, l.92. 208,22$-,24Q5672-;288<, 3u'4, 320, 33o-32 38-, 38 or 400 mu' Ahcruauvdx tile £v r' in co 8xfiOK 4 S(K_ 2\400K I \ I \1 tornui In ot Ki mstanc Ic ut a 8'd ¶K 4'c44K xlOSK, 1x244r totnwt 1 O2.17 The array can comprise a singi.e arra,y: The sth.g:Icarra can be aria si:h.gie substrató. .4.fternatively. the brraV is on.mt4ti.pie tdstite.. The athi can eompdse:rftrJe L' rnTh, Tne a a 4ii cOu1aflS.' a a a un Th ik mm) ul a i a cdl ompi ac LO c müt.ata' Fc> c saiiptc the pk Juti otrry' e 4n COrnp"i\C a.. 1e,t ahkn 2.. 3 3,5, 4C1 10.11, 12. 13, 14, 1.5. 16, ii. 18. 19,20. 25, 30. 3 4045 50. S5 60.fYS 70. 75.
80. .85, 90, 95 at 100 anax,s In sonic i.n.stanc'ot, at kast.two arrays of the piurahtv of altws are at least two arrays ph.wShty of nu's:.ath different, 1002 1 81 in smite hi sU"ue's the an.aV.comp:nses s>nnmecrieai: eharnbentt areas.
eanpk, the army: COifl)fl5C5 Q:,5 x Q,:5 nim. I 2: ni x rum. 3.x.m, 3.5 *x 3.:s ii urn. 4.x4 mm. 4,5. 4:5 mm 1: x 5 nun. 5,5 5.5 mm, 6:x 6:t*m, 65\65u1fl 7 Cmm 75,75mtn,8\8n'm 85x85mun'J\Omur 9 \5mnt10 x.10 uho, 105 x [0.5 torn. U xli i'S 11 H.5 Unm 12 x 12 rt1tn. 125 x 12.5 mth, 3 x I mm, fl..x fl mm. 14x l.4mm 14.5 x 14.5.mm 15 x 15mm. 15.5.15.5mm, 16 x L6 mia,o\1o)ninz>'xUmm Nx1/5mnL18Umrn,'$5l\) n,'9 mm. " S. 1° 5 mm o 20 x 20 nun cami eta ues In some nisLirce', Inc anax ,,es 6 6 5 mm hamhereJ ai N Alte' uatrt sift i'c.uu a comp"ie' a'v nmerr cal di inlxied areas Foi exunupIe the aurA compnse' 6 5 x 0 5 am, 6 5 " I tam n x 5 lilT, 6,5 x 2 mm..6.S x 2,5 two, 6.5.x 3 mm. 6,5 x 3.5. mm, 6,5: x4 mm, 6.5 x 4.5' into, 6.5 y5 mm, 6.5.x 5,5.rm'ti. 65:a 6 uirm6.:5 6.5 nun. fi' x 7 mm. 6:5 75 nun. 6.5.> 8 Thm. :6.5r.,.
lOnto b5',Icn'un (sSkH nu' 65xL:rmr 6,5c 12 rnm,.6:5 x 12.5 mum 6.5x 1.3 mm; 6,5 x'135 aiim f5 14 mum 5 x'145 niim 6-5 x thm, 6.5 155.nthi. 65 x 5 x.165 mm, 6.5 x 17mm. 6.5 x i7.5.i.vum 6 18 mm. 65 x 18 5 mm.. 6.5 x 19 mm, 6.5 x 19.5 mm, or 6.> 20mm. chambered areas,.
OU2 9j I i' aray d11 ou'i sc 4t icas $t301u I rn 2 tm 3 urn 4 inn, 5 ttu ( x:i 7 4111 S im urn. is Ufli) ifl 2() ni 5 am. O ni, Luli 10 nr I) in, ?U. in 5 ttp 0 ttr (5 &m 70 ini 7 nn X() S uri fl an, 9S t 100 m, 25 i in io urn, 75 Lt D 200 itn 22 rn. 250 an. 75 am CO urn. i2 pm. 350 pni. t5 m 400 m 425* tm 45 um 475 tud ar 500 rn ats: iii saiñe instanëea,. th at'r corupdses 70. uit spots' jffl2 20J T' *r OTW c lfl (Oflif'i F 4 least -$MRH I ta, 2 urn, 3 pm 1 put S urn 6 l in. 7 a 8 pn, pin, 10 u.rn iS pm., 20 p.m. 25 urn, 0 1irn, 35jn. 40 p:b. 45. rrnr50pnr,.55 ,tnt ?0 an. 6,trn 70 in, 75 urn, 0 pin 85 urn 0 pm, 95 100 LUlL 125 urn, La) urn, urn 209 urn, 225 urn 2S0 uc, 27S urn kt) urn, 325 nat 50 ni, 375 m, 40() Lrn 425 uni, 45.0 am, 47.5 run, .500 gin., 525.um, 550 urn. 573. gin, 600 urn 625 pin, 650 pro, am 700 pot 725 rua, 750 urn. run $00 pm, 825 pm, 50 pm. 5 urn, 900 am, 925 pm. 950 gin, .975 pm,. 1000 m feature. ptchJn on distanee. the array compfics 161 pm icature pitch {00221! I.w ra can coinpuse one oi mole orobe's lii some ln\tanc:% the aua eomptLces at least about'S 10.15. 20, 25. 30, 40,50,60, 70. 80. 90, or 100 probes.
Ahernativelv; the aims' eompdscs at. least about 2.00, 300, 400. 500, 600. 790, 800. 900., 1000. flO0 L00 I 300. .400, 1500 1600, 1100, S00. 1900 2000 200 2200 2>00 2470 250, 2n00 2700 2890, 2)00. or 3000 prob 1 he,irr,.w ca tomprisi. at Last rbou sUU 4000, 4500, 5.99(1. 5'0fl 6000, (>5(10, 7000,. 7500$ffi)9 5flØ 0000 95) or 10000 1ihes In,umt >nsLrn s, ti e % ontpi s. 4 kas{ about %u plo>7s \ i "flat Ret'>' I h, at con3p.r7sesatLeast about 2750 pitbes Tie probes can be spcd±k.fo the piurahty ob olig90udcdt.ide thgs. The ii6bes can be p60ifk for at least a portion, of the.phjraliy Of oli.gcamciet tale tajo,. The probes can be specific fhr at 1act about 5%. 10%, 20%.3 0%, 40%.
50%. eO% 7 0% 90" >, > 7' ot 100% of the total uumhu of thr p.i raht' of ohonudeoude Laos. ..Mternathe.1y. the probes are specific fOr at least about 5%.. 0%, 20%, 30%, 40%, 50%. 60%. 70% 0%. 90%, 95%.< 91% or 1.09% of the total number of different oh&onLL Jeotale t s of the p1uraht at ohgonu&eoude t s In a her 0 me', th pi oOcs tre *ncn,,pecifle probes. Forex'ample, the.prcthes can he specific.thr a detectable label that s.
attached. to the kihe1ea$noecuie,' The probe "tin}e stitptax kthi, 100222f The array' cito be a prhited ar. th jty in.41a.nces., the rhtedahay eotnpcia6'S one or more o gonucleotides attached ttra subPrute, For example, the.printed array ecrirn ses S armoc moLr ed oagoinccoiidcs ataithed to di) Cpo\) slictre >d1btTclt {002.23J lernatji;.d.y, the array earnrrises a slide with one or more wails. The slide can "F', :cempt4eJátaSuitj,4,5. t;4% 10,11, iziZ:t4 154:]& it tt 19.2% *:M# 35,4% 45,50,55,60,65,70, ?5 SO, S5, * 95,or 100 wells. Altatiatiwly, tM slide IZA. * t121.. $*.14P1% aea..
85Q090O4:9O,or1flO0 iSancs hcistI*coø tw& K. *W. 96 kT ot ts:4!=a i%, 141tflU.4Ut 4*; ii4. lao. s.; Sar.* wok f00224j Iatht*3niars:tf1j s*ppo wgn A05 SiS*flkta)ç AErqj&Lrtdi 14 aft: cbnkMlst* tt cOfltfl flfltCt,. npat printer, dot printer, orpn primer {*fl25 Tht *lid *ippott cati comprise t use aftscsds IS sit as*enxbk En mkrowefts thotlidippapEisos A tt4i tohtSg s:ApEi Miroerray n*x1P1ci' syt [$226j, Itoth waat tIni1d ap iSavbóe Iflarpie otpiaMsinctSv,. but ate nor thnttcd to, tvj$I) nmtttcrty plates, MW Mtdt$pot' platts,. micnpla P%uteO4 umicrnplate,MtaPlaMMELFIA rita %thPla and LiW; ti i:Ens tomi si aø::. ss: oi: o iprà &annymetIudeSanpt ljq. i1jases, veSrsse p*tyr*bt*set4 and tjtt4kith o:ft *. StteL Sókk:ftke oOtuIçi Efl#$*::otflEtISfl$Ifl4, nSA ujase i,:bi9A gbüI bAS1t and T4:A Md*tWjiSijJ uT4nikliga'ezan4 ARëL Ion,]: thinathakhs, S sysStcdSksS hercSan Shenomprisc the u:ot one or more ttverso transcriptasta 1t som Stances, the tev*st transc4iptaae i a MW-I se:t** MIM1Wttv* tsaost AMY stttta, : tdontrase reverse iranr &1tsonwSsances,the:retrsc fr user b i:1⁄4MW SE* :s$s t2i U*Oone4t mo*LE*m*iOWMr44raoos*fl*d%bUt*flOtióithdtD nl,ThNApO1yrneaadi; PNAo4y rasc 10 ]V. Commercially available DNA poiyxmsrasa bcbidc, but uc not lunited to, Rd tO t)NA PoatttzOWarrn$S:s*Olymn:sflNAyob,, &rWilóbust$A 4$ PQi) TrersL. LV. Tac DNA. Póhrn.ei:ase. 9 N 1'kn. DNA PoIyrn.erase4:Dee1 VenRJ' (exo) D\ A Pohn,ca%e Dxp \ CrAP) D A PoIvmi ase I lerno kier. L &q Loni \mp D\A Pot) ieae Urn DNA tkvmL1I%c Phw t,n(' 1)\A Pn1vncta. Q5" H :idclitv:DNA Potyincras. JhrmiintoiM < ONIt\ 1ohmcrasca therniinator DNA * Tharñinát&T.u DNA PoIvmè n sc. fill DNA IPoiyrnéra:sc, VntP D\ \ P01) d en'c VLOLR (esn) DN \ HVLflC14'A thu UN A Poyrretase phLQ D\A olyme sc, Th ON P yn1naLe T7 DNA Pol nwar. ant lc'rnur<d T;jnserp,e rc,c tir poR'nu a',. n RN \ mohnti >N" anti a' RN \ p'wit a'a 1, RN \ pOTh4' II RNA po!yiou\e lfl E ott 1totv(A çolyncta'c ph RN A polvmenc (RdftP), ictlyW) SP& RN p&ymcrai, and 17 RN.;\ pOiyTherase,.
{O23q hi sonic. instances, the. rnethods,..kits. and systems disclosed. hereto coniptise, one or more: restriction enxyrn.esJtestridt.iun cnz rues include type L.typ.c. LL type. Ill, and type. IV restriction enzymes. In some.instt.nces, I vpe 1. enzymes are complex:, mu Rilsiibueit, combmanor rcstricuona.nemodtcaton otzymeij that cut DNA at rain cm fin from their.
recogniion.senuences4 Geoertd ty tynt. II enzymes cut DNA at defiPed positk.ns c1osc to r *wtiun. their reco onion sequences. [hey. can produce discrete restriction fragments and.
distinct gel banding patterns. vn lilt cnz.yTheS are also l.Stg;C Ctfflt3tTht5ll1:rCStti.Cti[3Ti4.fldr modifica.tioii enzvnes. They often cleave outside of their rcco8nJ0011 sequiccsind dan rcannre two such Sequences %n. opposite onentations.with tithe sar.n.o. DNA. tti&ecnl. to aecnipI.ish ckrvge; they tar&y give comp1et dgats In omo i.ncthy type IV eynics tecogp.iz.e mod.i ted, typical lv ni th.yhttcd DN.A:and can h teamtified.Ihy the McrBCi. aid lVlrr systems olE r:ofj 1OU2$2 ti 11iseelhmeous (tunpo cuts Oft:.i]'he. methods, kit. and syciems disc,se.d herein can c.o.mprtse ie u5e nt one or mote cagcnk E'caniplc cii cugent' mcludc bui are not hnithc to PUR cagenh, h. tnoa reagents, reverse transcripti.oti reagents, erzsiite olagents, reagents, sam.pk.
ptepawtion reagentsk and. reagents: ft nucleic acid purification anti/or isolation.
[00234J The mOthods. kits, and sstems disclosed herein can cortiprise the use alone or jflfl huttenc Examples: oibulfdrs:in.&rlude. but nrc. not i.in.ited to, wash. butlers, ligation bnnffn rs. fridivaiion lu.ffe.rs, nnip.hfieariou buffcrs and iec'er.so transciipticnn. buftbrs, in: omc instaices the hs'ht dnzauen huffe ts a corn uct. ia Its aatL he buffci, aicb th M \C Ilyb so.hntioo, SSPEhybridization solution, and. ECONO hdinidization bttfr. The bafflers d.isclktscd herein: can dothprise one or rbure det&ents.
{1M12.35J The methods, kits,, and. siystcmsd.isciosed herein can comprise the useofoneo:r niorcankts. Cant a ean exihasceerimpmve Ike effickneyof:one or nmntu*cZiGns &hSkfl4b( tIt&sS*v;'utti a T4a::$:sj 0.44 Set:or$kp&,:uiea$r 04. te1S,rnfrc W,ol-tG$Amokttu1; WdapiicoáPotx*ktta.icr kb1$:i,:k, S.;T b::,iSt$ ars.carteasantttt cfthe ns.kcut be SJaanypo4ucithacafto a uronuS(tg; n;eppeodSpt'4);Aktiaiv4t)wcatti*rcatt *i,zktS th c fl,: n!aStSk i' s*:,ot:t*,oc* bca4 array, Øass, sUdc, th) Carncns can protect t m&ccute at any product thereof from degradation. For eXatUj)lta C8ITLCt$ C6t) protect an RNA ño1ecu1e or ny pSnct theft&fmx tn1scsMtiSly4,csqmpcote afl$A iS,cnknw.anoductihcrqt' froth'a DNas,. Exam sotctIonilad S' not ith'Sflnu c ad th"Isuici $ii,b * ,$4,..a4d/t ti.:.,Wyi"c*i4i Ltj4$; ptIyadcny1atectRNA, phase tNA, phage MS2 BNAEak &NA, yeast RNA, yssttRN& :mamnt$*flflRnnngfrg tlM, sbort pøty ditylatethi,h,ti': dbo*mclóa'Mos anti :k*j'sciàs t*. kiAt' kt,:t'' k p__f R$k A tWA cattier cati be a. non-polyadenytated R'NA. hi some instaa the earner is iftun a bacieciz, yeas4 o.F.'campk4h.'eante i*vvbta nuttók Sd ttkic4twdtba yen Pa: eto1fr *aSr a:prSn hm 2adllha In O à ci'tSJ$ká$ front ccML Attctna' t&.cant' ka atscl&S' Sccrá Cr pc* tamamm*1.
s:": i:,:s t'':nSs ot't.
tO23tf thta*ttho&Mits, S sysietacdStised hrcSanonpdtthtttstoEettor morn tontsol agcnts. Control acnts an bte1ud ontwt oligo; inactive cnzyáxc's,, non- *.$MMitivtha'1:a"to.Spthio ",h'4ñ,Wi' prntre:ceSy c.athd,tonsosk4ilce4nlem$aPCR anipktSthn couSS; Tbc rc,'ss'*ø, *:t",' $iittØIs. I4:P".'.SflI.
Th:nnci&.acid tn$ contrnianW cf ratioi tbe' u1agents a ttmpthett or'ttsttbbct& E(.:',U $pulfl p1flp$$ 4atbetbSpjAtettMi3P".,added a:"ci44isj1pjj P C%alnt asp*tk',tthtØ". :mfl be *Adoi h M *AtØSSreWt"e tt pMn tunpW to bt," "ìb S' S'I "kiwt':,',:i" .: ttp:,dannkitit a :g,,Ka,tht.gflkc4flfljtatG indded tioLaSw t; 4, 0, l 4U (yh, l2 Ii- : 3& 400 an)phtcatto1. cyck. ite "** hi tetpIate cai be added to the. n1pifi.c.tihn re.a:c.tiOn 1hy flflh b tot.. i 1t a im]ifk tôL eci: 1 ke-ui enkate can cttnp i e on. n 1Oit.
nueicotiks or midek acid basz paiis. [he sçilkcHn enipiate can eo.rnpdse DNA, KNA., or any cmbThation. t.h.erc.9t The.spilii.n tet.r1plae nni compdse:*o 5r iiore* hbé:, iOft2.3*J TUbe methods. kits. aid.i.*tcm.s * disclosed hereit can co.npr*se. the:us;c ofone or 101 e pret ip and o cOn abe' v<ik niuinx dU plaics) In o no i a iru as fie 1'npt. tip ice o; Thad NN ups \heitand or iaduan afly th ont ieltrs in K' Ion binthug contauicr\ ox hirdin% pipet nn' nd 1os brnctng Lont4uior nun ha'c redukd eac ung avUu1 subsecporn inipk dOi tdtt20fl aynctitetI n nh siLon4 ad p id non owbinchng containers. Low binding 1ipet tips and low binding eqntainçrs can havo red ced sam do huducc cor'pared to nom on binding pipol ups and Lonnnei s LxarnpL of Ion hindm ups inc ude. hut ore not hninod to C nail w'' Deek\kotks kin Ii odiru tips and S\ cWt Pi era ant lan brad og L idwu,d cps A ron-limit in',t lt,i i -Ii mida corn lUll I inciud* Corning® Costar* ow bkding microctntrifhge tuhs and Losmobrand:ov binding PCR uibcs and microcentrifluze tbe& [00239J Xi ft hidca1iofts [002401 The methods dse1osed* heitti a may he used* in gene eprSsion thonitoring.
U last i ip pi ofil ip IiIir<a"y ut crnn c nolypiup. cn1cliet anay% s ml. wi-It ion pattecn anaIs'', tunini tpiq, phaim in enornrs any;neucs, pathogen r n9iin and detection and uia fl t,s tjs,ii \p'l, 5Sj[i] tfl0flltQYtfl' aid pio H n& nethod, h rs-e been hon a in U S Patent \os ti 0L44°, 6020 L 6 Qfl M) 6,040 i * 6j77 24S are 6 0° S22 GcInotyØiag and, uses therelire ate thien1n di US. Patent Publication Nbs.* 20030O3iO49 ariç Q 700oSM Jo and I. s Patoiu. Nos S 092 mci oo SYSji59 o 2K4 4o0 A 6 1 947 9 irn' fi 353 179 Oth,.r us',s ate nbodied in L S PatCfll Nos 5,O1 92kç 5 902,72.
6045,$$&, 5,54:L96i. and 6. 1 97,506.
[002411 Disclosed herein are methanEs, kits and: compositions liar detection, rnomtonng.* and/or ptugnois cIJa diseaw or conditiod in a subject. enerallv, th. thcthodcompniscs (al swchasticallv labelirna mokenic to pioth cc a stoehasiica.iIyabeicd moiccide; and (Ii) detecting arid/or quanutyrag the: steel asncaJ); iabekthno:iecuh,' theteb detoc;nna.
mo'utoung indoi plug"osing a dNeae oi conditon ru ub ect L1ctectLl dlM).tSc o LenuLflen Cnfl coinOnsO diagno'ana ad \PO er ondflttin 1002421 Mon.itoridg a di eits.e or coridhiola in ii subject Cdii further komprisc Tnon.itormg ii rhorapeunc tea aten Monitoiiao a thetapeutic rcajnieu can compuse det;immtna, inc efneoy of*a tbctapeutjc re:girneE. tn. soTne irEan.ces tnoni.to*dng th'nipeuEk regirnei. on1,rise$ adrninitrang telmlfldtulg addn'. iriknu a ther1ncuc rtntn ltrn. * therap:utic.
ic'lrm4 i In cti pi e u cMs IJ ut ChLC1lir lIE dot dn'm6 nccae icy. r mode r a.. dmhi istration o f a th&ap uic rcgimeii. A tiw.rape.uic rcirne:o cii compris.onc or mt)tc tImnipeuti,. - Thctheapeutic ditgs em be aia atkauc.r drtg. ajtifra: autihaeteri.a[ drug. autipathogc.nic drug, or an:v.comwnaEion tereot, j002$3j 4 Cdtcer t!O2$4] Lu ornc intances the disease or condi mu is a cancer. The molecules th be stodhast.kdlly. labeled ean be fhym. a caticerdUs cll or dssue; lit sonic instances,, the. cancer is a.jC(,fl a, catcmomm. lyin Thon ant Ice a alcnnias aie cancet' ott Ia bone, eattla;e, fbi, muscle, blood vessels, or other cornect.ive or supportive tissue. Sarcomas (nelude, buL are not limited to bone cancer. fihrosarcoina, chondresarcorna, Ewipgs sarcoma., malignant henia.ngk)eido.theljona. lignart 5flfl0Th bilateral vestfteflar sch:wannoma, os.t:.sarconra.. soft tissue: .saeonws (e.g. alveolar soft pad sarcoma., .aagio.sai'corna, etou'co'ua ihilo'id:,, detmatoF Mfl'cOTfl dcnoid tumot, cmtie oicI srcorna extraskeictal:osteosarcctma, ti:brosarcomu, hemangioper.icytoma, hemangiosarcoma, Kaposis Qflfl* loin fliytms.attorrta. iiposarcintma, Iytnphan.gio coma,. Ly:ul'iosarcurma. irua.iignatit fihiO,,p, bISLOCVtOnl4 acu. afi'wtcorna rh 1odo.nyosei coma, and SV.lvu 11 arcomal 10(12451 Carcinomas.arc tanuers that begin nn.he.epithchai cults, which are cciLsthat cOVOi' the. ;stirfhce of the L)dy. produce hormneE, and matte up glands. By way of nor -iththing cx in pIe, &a CuinitbiS IL de hrea',i cdl pan& I aaQc & cV i lung c.n& ci * color & .nc e' colo.ieetai ca fleet, utetal cancer, kidn.ev.cancer, b ladder car.:ecr stomach car Oct. prostate caficer liver cancer, ofariari eKe., brain cancer, vaginal cancer. sulvar cancer. ttcri.Oe cancer. nra.l cancer. penile cancer, teajicular caner. esophageal cancer, ski:n.eancer, cancer cii the fallepuJi tube', haad and nck c mer $StiOfti est.nal, ion4 ac flç'i,l,hilflOLUtL9Olt cutaneous or!Titraocular melanoma, cancer of the. anal region.. cancer of die small inttstitie.
cancer of the endocrine, system, cancer of the thyroid land cancer of'tli:e parathyroid gland.
cancer of' the adrenal cl.and caheer of th rethi cancer of the renal pelvis, cancer of the ureter; cancer of the eadoma'tnuni, cancer of the cervix cancer of thepitunary gland.
neoplasrn.s of the. central nervous (CNSJ. prima' (TNS IyltqThLfttIa, bniia. stem gikunu, and spint asia twror$. hi some inStances., the cancer is a skin eafleer, such as a basal cdl carcinorn squamous, melanoma, ttonmelanoma. or uctinic (solar) kerato,sis, {11024t,l In omc inu ice', the e4flci i a.un$ C. nicer I ur g cancer can sLu Lu' the airways that branch offthe trachea to supply the lungs. (hronchii or the small air sacs: of the.
kug (the ai.veol.iL]1;u.ng cancers nciude no.tsrnaiiH e:i kw.g*.*caTci.rie*uMt (NS(I:Ci sina.I. ecU.
king caitinonia., and meothLiomkL Ea npEes of NSCLC.th.clude squ.ntts cdi careirttm, ideoate.u:orna, irtd. hirge. eU Caremoina.. The.mtsathe1kmh may b a e.ancetóu.S tunTO Of the lining, of the lung and. chest cavkity.(pic.ura) or lining of the. abdomen (pcritoueum)., th'sotheiioma niay be dub lb asbbsk,'s bposure The bartcü maybe a. brain cthben. such.
ghoblaatoma, 1002471.Ahernatoiei.y the caneet' may be a' cent.ra ner:uus.svsteen. (CNS) tutnor, CNS tui IiOF hC "d.\' I lCd f honia. UT iIUfl'lltllfl I i lKPlid I 14) CC Ud h'ncifl Lie i grode glioma difThse lInrln\lc pontwe g.honia E..',ampks of gliemus inC a' to.yto mi' oIigd dtoinm oi n ixw c of ci denthoshny a..nd a>tucvtunia clementb and ep.cridymonias. .Astrocytoma u'idu.dc,.hut are pi Umited. lo low-erade atrocytorna.s. anaplastic astrocytomas, gi.iob.k stoma muh.itbrme.. pi.ocytic asttocytoma, pconio ph; a t olhPOcs1oflis, aud.u erenh ma giant e&. in ocytonia flhg&1cndlo<Loino\ n lud. kn -.y o1l&o.Lndn\g omt, or oIi2pa.Uoe\ bn aa) 111U anaplasuc oUguoendnoJon1a ?Jonghoma mckde Ir vngioma, Ntunary adcnomas, prim.arv'CS vmphomas and medLdlcib.i.astonias, in some instance, the.can.cer isa i.n.tii'nglotna..
[0024*fl The icji.kc&da. may be an. wiite 1ymphoytic iehkenha. acute myc.locytie leukotnia.
hromt k a plait, a. kws.cnra UI.. hiona.. m cu yhe I tria Addat slIs.J nip..'. of leukenias inciude hairy ecU lekeaiia, chrome niye1omonbytie kuketth,. and. iuvnilè niyekp'l (itiOLS a. Lux..ni I i00249 L..ymçhomas.u: sncI-ot tu1e hriphocytc\ and AThPI Je' clap Ton Luher Li or I Nnphotvw,s. be two i.naor types at iyinphorua ae Hodgkin. lymphorna, pIuvlous.i 1-. iow H ion'L' . do and. non-HoduknI\ R inhot ia odakin rnphtv'n 0. naf.c'e by the pt:sna.c at ñc ke,d-Stcr&xrg cI1 No i-HocgKn' Iaipl oma arc afl lrpho'mis wnjc.h are not.Hadghin si iympho'ma. Non-Hodgkin iymhnias may be irSicrut iympl.o.mas and agressive lvnhphomasL.Non$iod.kin s lymphoma.s include. bat are'not hmitcd'tcu e'ifthse.iargc B cU iYWhomh to.1.hcu!ar I.ymph.oraa rn.Ucosa-assoc.iated. lymphanc tissue i mphoma \L\i) na..dll Isrif Ioc'. he iyunphouu mantle (_cil lymphcraw, Bud itt.
) inpho ia ne u an anl huge fl cU fvniphorn W.ddonsnnni inr.iiohulmr inn r odal inhtg.i'nAI.ZO!ic U cell Iymp.hOn)a CNMZL).. tP'kflt maidt'ai.i 2k)).C' lyntphoma (S'MZL) e:xtraaodai marginal zOne cli iymnhoni.;. iiitravas.cu aT lame B coil ly,niphoma, primiary c liisoe {vnphoma and iyniphoTndtold gt ITU tm18tO'ka 1002.501 E. Pari:c&genic Thfuction: Oft25 ljIft.s oi.nc hi.tiricstht disease ct.co.r&di.(ion i a p:()ecij in.t)xik,a fle thokttik4 to.b t nasticallv j:ahekd ern b' fitrn pathQi. ibO pat.hen cn bat.ériini. p'oiozeatL. In 1I)Jthnees. {h:pte..: n y l.c. . *rfózón, such. a t( cntha,iu& ba Ic f r on o&liaini u1! ,r t. I /ar< h* A... ;-hicbde, 4 **heah't A. dh>onflsLL blUChOk1 (g, * (vnnori. i1 vesjcujcflumJ.
( :z' \1)rhu1n V C C c. ii) S] (ra & t t i /thh Sti.èìì< t/'(n Ic t; 1' urc. igh r. i/i. W / it(7stU/V Fvct»=uebi ( ; P t Pfl (, t Y3t}1 Ic bete.t; (thtii 1e5. C /tnbh.i. !por c, / bc//u mn e tf &27. aflum, %/ & ç, V TtI.ic) \onw C \ <iea V 1/OflPh2, P/e tOOhOtvi, It (hipkLvtophora:nt/0iih 7..hcnnIkA.. and.
Vt *2CGc/ 1 ii: pdthogen iiay h < 1 jn. , uc <1,c ( i ptu4 Ut (V IfiVtUfF1QUfl5 Pfl4Thfl< f) St dnd ta /notn St 10.02521 The. pathoen çIr. he bttct.etjum.. Exempbvrv h crerid incline, hut are act flnijted hr Ro c letLi Roi n. u, Br h - k to Cli.iautycl ia, (iir.dpb da, C' os ndwn C:o.rync:bectetIutn, E.nvrococcus, Eschcrichia, Francise1la. .Uaemop.huius; Hdicobacter IliegioneUa, Leptospira, Listcd:a. Mycobacterium, Mycopiasma. Neisseria. Pseudomonas, itet:LSia Salina idHa S.huigelia. Stanh:vkcocLus. StreikStccccus, in. ponlenuL. Vibrin. ot Yersudà..
0ft25.t]he virus can ix a reverse tr set bina virus. Examples of reverse trauscribbig v.iruses.itc[ude, but are not limited t, singi st.tndd RNA-RF ($sRNA-RT) Sims:. and t'n bk -orand d DN \ -R r (dsDN 2\.R a-us Non-hrnfl u:xa rpk's of &R \ R U su usn ib.c u:d.e rc.trovnusti:.s, aphare.tmvirus. betaretrovir.tOL ganunarctrov us de1t.aretrovirus epsUonretntvuusc Ientrvirus, utna va-us. rnetaviri.rus, aM rsc.udovu:uses. Non.-iiniitIing exampLes of dsDNA4kIF viruses include. hepadenovirus and ca.ui:movm;s. A.iterrutt.iv&y the.
virus i a flNA virus or RNA. virus The DNA. virus: can be. a duuble-strandc.d DNA (dsbNA) v m' In etne tusta icy. th dsYN A vu u' ts an denovtru herpes sit i. or vu u Exumpks of adenovimses.incht:dc. but are not flouted to adenovirus and infectious canine patiOs snus Lamres or herpe. S rusts mcIuLc, but cue rot hrntcd o hcn.s s virus, varice.lia-zostcr.vinp. cvtome:a.kiSlflls. and EpsteinBarr virus, A. non-limiting listof su usc', fl( !ucjCs siiudjpix S II U, C'tSt%S pox S iflb. NhC'.?p pOft. S Ii US nflt)\C, pus S JI5 ind sn us the ON \ s rus en' be a snut e stiandect DNA &ssDN c nus I he,sDNA virus can be a p.arvov.irus Exampks of parvovinises include. bu are.not. i:intili$ to, J 13 CdUtlflC p'k 5 virus fllOu',C 1di ci iiC CLU SOvIflV. Mine panieukopcna, and Mink enterif is virus, t$Si: A4Snativdjt vfrai aRM iiu& tkA ts: can be a dSb*raaded +j an'kSA t(( A)tsA orthoreovirus tyjxwfrus mta*ua, btue*ongue virus, aM phvtorwvirua. Examples ot(t; but u*thtIMtid *t4i:;. E*Ethft$& piomat, ñc1ude, buite not hiSto.;ótrot rhIxnvinni h4pakwki, eSovS;*S%4 jlioñus paztththt4i C * bttvitu&tSchoYfrut, unt iigaüMñubu1tcit!*M eitctpha!kis virus, Wc4icm equine tat epbaliti ritus, Wnenielan equfttt cncthalks virus.
R& River vb (Th$ttgtnyong t, Cbkungw%ya, or gebilt Jbrpst %*uS A txn-timit Log s::ofEsvA:vk:s4aynvs4 ditkv1n*tsamj*d otnyxsi4inudott arojotiSSt, &izit *n: Sn 4totsm; S: T: fK4MiS, i::Wi$o, bmitedottThm,4cShsbdo'tnephemetonws, tyssaiMi *MthsbdpviLjIs, tt** j C.g1f44S,*,, teOi$i,I herein can cornpria diagnosing a tttat conthuon ía a ptgtant subject. The methods 4e4Ihwdkscpthddiãita1èaflibn*smSTit Md*$á*$y th tboi4b*4 S:W M [OOZSfl Thmetltds*li: a s 4S SS b4 in th i&s Trisomy J5, 16,or 22). In ether Oases, the trIsoty that is detected is a tiveboth tris*xnythat rn:tm itwaMsyndremej,'S tr1somy:i (DewnSyi14rons) the :f P1*4Wt*:ø*ã*.$S(*&.
(Jacobs tdrcn4:or)%t(Troi*j 1:molecuic(S)t abckd, onsov ne*ft&Ungttoniotrhs: k3 lXj2JXq ott Thr anipt4 the mG ecik son du*k*dkron*emcwme 38aud,konchrAmçsdme 13 10025k ISicaSftttas ih an bcte d on*n1,o4, Idt S systems disc bied bcrem include tmaosomy alone or mere dromosoun (K tmosezmt rnOZgNSOmy ahGknOWn8*1uiflefS*5STOfl1e)ttnSOmyOfOfleOrrnorecbteflDsGflW5(LiL i,a4)ç$,iotnsony;at*J peraasomy:gf*ne or. edwmmes(wkichln swnansi most flttbfit)dbs: xib,th1d"tA:w :UXU, :i,flfl,XYflad si' totnion, 69ttovne fl:* $ta.:$)+ a aM:ftWdy.
EXEMPLARY EMBODIMENTS
tVe*1 Pisàscd heroin, :M:irn takdiasn aenwthds aqSSs stt5tr;i$ioxtots:1NA:mokte. laso tthpiS p4Uöi1b4NAi1*i'**ii unleatics with a plurality ofoligoxuwleotide tags wherein (I) the plurality ofKNA uwk'cuks (0) dtent uttiqu identifier sEquencec (br) conducting a frt nd syntheth, nactS by sm::$1hii tbh'1*RN$*ltWøflfl *tc** bi*::tan zip: pt$t!.fl iaSJSkDP4A moiecuk, and Si4a:SA nktoULstpptt, tW2601 Pi'thting:a 4iipdtLN: SuaS ati44nü 4iókiø1 fss4hA: flt it6 tts nttSnv ii adS wthiRJA tiby rnc4ècutà by ligatha Thc*tsthrneutetthcoUgonuthttideaa:cornpr*tustoft iw ó1; Tho*onublsfkk t$ca eEftE1*d:to an$thonoftheRASIot :F&s**tk, a 1tiiidE tt Ø&bó 4 to t1t5':ikid fTho1N4 AliSbcthetionStSi& ttafl: attaciSi o: tht 3IeitdGf kl4iniIecuk tnSttr WsSSPiaiThø:*::stWT:n flflAni.Ø., Meftntnt;ott:dligoimcbot3&tagtQ Th Rfl&uwtwulq $fl th ua tUte S4pôtSktu1s 10*1:1: a:ts**:as**a:j&te fl targix:ecffic ron can Mbi Stnt ohhe plinLity oSgwwcS&i*taiiai ..e:S1tei4&spfl:tSIe:ae*øS p1u$tt InaiSanccs:thetarg,.
&rgiatit1b1s aftadth óftheplut hyd at Mc ufl:b'jh*t:: ;;apoifie $ Ethblth ttiicliñS dtM pbinltj. @4 U tue Si t%agst at teSabosit. tt*ortd &ftii mo1ècutis kwoSSSces,ttargá ckfitvrcgknenabi attSbisntutthpwøkyof fjgjfl4:g to tS::aboutiii2 13 1,s 15 dtftbrecM sAnss:th ati Sztofthe KNA:nkcttk, rwii, ** .$ti*c, 1 tIde $üThbnSài 4 :xgOn; The mf eèiàankeptaS SnikSsat pdinerregiaat 4figj Thjmudexaganat:ntuSotiIe;tniSgrh: Th:4..* :::* :$t$øn, Watt:. fl,ioé*14o tS$c4fl eethpri e * tM.anth*. wa4d t pSJaisentoz pnjc}eqj par: a:t:*::: II Mq$**ftth, dó 4 4NTP mh, anneøtlng butTer, liga 1iation buffir4 or any coinbintbn thereof Conducting the first stran4 syi*hcsis reaction oan figther cemprLs a first strand buttcr dithiothrsot urfl, KNase bUtiS; bNA:pO1flSl:orcoflibia4tàt1he1tof t002641 tcfi;s4snthSsreattininafut pSathsfl st1 Sst strand synthevsseacuon can btr campnse a therinl eyckr program comprising 1 cyde :f5tfrO ntthibwtiitd$°Ctbr2Siwiss,$tCbninsan4.
°:2Ei 4t iiy 1, c ie%4t4? Cfotat1at Thtffi'S *S4 hâ'i c* tt4her jti*tS *0 D$* $ai s(hA*' sp*ói&ptt.t pe1&tht t*,tlbn: 1itotraIitiaflk'NA printtktst specific pnnier tan bybrfdxze to the IzbóLed-cDNA mcøevuk ai4 a polymerase chaw Teagt)n tan konS:Sqtubatbd::iAatecS 19026S1 The ssrtplc caub ftxrther tested with ont ornxw enzyittts to rtnaw or deradc RPM imtàósk$ fri, :ndr:iur &(thh*wt w MtaniatüS.c t:MinpinnóS dwiftittSflM yc:yii. :J$)zo h$rolyz4 titMttSA [$2.1: Th:j$tip4i*1M*W *e::4P igj pIt%ni$*::ti(Pz t tSoc lhtd-smólitiarneStAncts,t p4rasedbabtactbmhaictS: *s: SrandedlabekMDNA rnpfrcuk:wkhe. tis:pck:mixse comprisfrg atM tatgM:sJ*c:wte PCR pi nier. urn eta PC ft p inu &vmu a2 bufiti D\ \ v° mu a,c d\T1> flul\ or coPibinttion thereof Itt flrt.PCR bi c(indu.ct*d In therrniI eyckr. T.t firt. PC.'R can cOtnIrise a theitnal cyc.Iet ptogra:m cCihprjiig I. cyck üt J4 (?: tøi2 üinu4c, tO(ff3%ktC bY $O cydcs of 94t ( thr 20 CCOfld.S. 58 C tb.20 cco.S.e:, * and 6S° C. fir () seconds. ibliow.d by I 1⁄4ycJ) oftS° C' oi 4 nrnutos and cck o 4 C f'cn t tcclS 2 nimutc5 The ne4cd PC ft CO1Tp:i.Isc ccrLth1cting a ac -and.PCR con1prisi.rg miing at Least a porticni of.th.c amphcons n the 1ra PC II ca non oh a sccmd PC P mnwre jnw,r sum A cxnnd ao y specific PCR. hmer, lahelcd-nnfv!saI..P(ilt pthner. polvmora.se. buffi DNA pnlytnerasc.
nitx or°v coc,binanon thereof The LL0flU argct spetthc pinner ran h\bncizc 104 rgiOii in the hs.hkd thokt.t.Ie that i$ downstrcim of th first target pe.cific 00cr... th& bed-universal PCR primer is llibel:ed. with a detectable la.bbi. In some instances, the labeiedurñversai PCR primer is a c:y3-iabded universal PCR primer. .Aiterriativcky; the 14,eied n:ei al P( i'& pnmct i'. a I \ %n i4ahekc at' e >al PCR p imet he econd Pt R Lal' l'c in thcrn,al b The seond PC R can ton pi i\C' a ihs,rnal ini kr pflILQ liii Jc o19 1-1 tot 2 minutc lol o%cd I) 30 ccb oi'14 C flu 20 scconth 5C C for 20 seconds;. and 68° C tirr 20 seconds,. thilowed by.] cycle. of 68? C thr4 m.jn:utcs.and 1 cvc:lc of 4° C Ilir at kast. 2 rninutcs The sucond PCIt.ot' thu. nested. PCR can produce a 0.bIbdani.plibot canrisi.ng ± moibcuie ol deoSe hrg and the dde tie iahcl. h. Smile instances, the Iabeied.cDNA roilceule of step ic is the aI4far;h on prodttced by the eeoi d Pta of the iteatzd PCIt.
1(1412671 in sium. uw{ c. s, dcie4. hog t a Lthck <bc D\ \ run teak onr'i c h'sIirid ing t least a cirtion of t.h sample comprising thefl hiheIcdampIicons comprising the eDNA thoiebu.ie, hhgohucicOt.ide tagand thb detectable tbbcl to ij solid FIyl*id.izlit at least a 1iirou ott! L \? mpk compi in tht Iahek'e-tmpla on-at' compri e a hyhi Ow thon mrdure eornpucing at Last a irflon o ilk \amp1e co.npnsing th. $xicd anpl'cons procm.ced in the second PCR. of nested PC., controi. ohgo. hybridization: buffer, or cnmhnation thereof The control. align can comprise thcdetectahie label conjugated to an oligohucleotide. The dbtectabL label is the. sie as the detectable label in the hthe.l.ed atupheon. For eaampk, the!ahekd-arnpuicon comprises a. Cy3 lahd and. the control Oligo.
cotntti;Se: Cy3 abided oiigomiel.coddc& The in. the hbrid.rizsticu.
mli:tqte tn denatuted. in ontc: it tat&i de.Patdrizg the.i4l.bi d4tmp kop.conq):tise$ fling the bvbndv4tTor mntuic at $< C In some ni>.,tane the kbndizrion ifiniurt a, icnhit,d at 95° C toi at tca>t about 1 2, 3 4_ui minutes <\fIct denatut aLon of he!abe1cd arnphcons, the hybridization tabdum is incubated, at 40 C liar at. least 2 minutes. .ibhridization if the kbelód mpl! At*Msqpfx)ltCSuGQEUpSGadèing at. kt:a4iemptthe h1widizatnMt ntt'uft to the oM sup$rL in sout {tthtAnces hybridization of the labeled-utwtll ntan AN twtayilid& flti 1d 1hscat,be Least Ubwzt 1,2. 3,4 5,6,7,849, 10. 11, 12.13, 14, 15, 163 17 it 194O. 21,22)23,24, 25t 2& 21 2$, *j, 3Z)LM **M *J9;4&4t.4Z4344; 4S It4t qt4W kbum Thtk diksuc ehóRtodb:tho:sttbr.4 b41⁄4d4ik.:bó bht$ *ti jita iut41tauivt ar*cou 1$. bflidized to the suppoit ft* aIsThtS-5 ht)u 445 b04*r4. 64 hours, 840 hoursi, 9 iIiurs,:i34fhoum,, 1:416 k,Ifl 174flo oit*Z4wnrs. HytrMiatin tthc Muot1ASfr4ft tatattitbottit*ihthdad *JUPL&COft and inQbattng itt JabøIed'amflcon ti4.supportst a hybridization t pntur& in entesa&kSd&rn4esptniuzdsit hSsbowCø21Yt 24t 25'' C, 26°C, ire, 289C2V1CiO'C.W ( 34 C, We, 3$OQ,4Qq c C$:D° c,ss, C, teOio*iI t1c *oiidsam$wt;ta. domp e aphtralityüFpmTh:phnHtotpobtai eo$:::: M$ô*i;k:P.t* dWlèøt :nlfror;kbc1Mirnp&on.thcpIura1tfltnbtcan:bennsttewthnoiid suppotiir ös saj: whet th 4atijon4e s Or øomp&e øuieasti::i O i**4 S swiAi: SiinS.s. t:óriS:dáS4::oi:tbnfl, trVtoti,, it:pfl.oapthi$ , s to1StS: :t spec of two ditflrnnt umqut identifier regions of the ohgwweleottde tag.
rUM thnttho&ttheronpSecowdng tarr fthawadlzSs* iopSn: t sated arniy sfi& The scaled arrayi4ide an be mtnbatcd at 3rC The sealed aitay slide cai b& bflaVVC óàiniS. I: I::à:bic* lSt$i 4iSütit Wc :tmove4tmch*eit. Th ybrtizsion Stacan.beSored lar C:. aanasoty,the itacaSiL.
[002701: Wag:tweftrcnpriS addbtg*Sb buftbr bThtwellandtn:Mpitathgtwth W4t ts: in some1nstanees, the antsy slidet scaoned4ry à g,fltd Snavcdthcwp1ki) dtdcacomps ntthampifith Th*t&:*h4 casbetedttauj9 flemSo4ócksedfherM can$,.
swuaciiiiitteS otIbtiSntxcu1S, bSnpth*not **søi::Moitfi 4t:d,: 14Wsi::bc IEabcSanptIo or hs ted4belcd amplltosprSto: Sathrneta oM molecuks, labeled.smpticotks, et framastc43⁄4bde4 anpitconr to tbe solid uppcnt MtSi, t1. dà. blq label 1 thi wUkbs14uw1cS anØnsor montS4ibtted a ko iaS bEthkdmo, labekLa'to : est: : ::w: :k4 ta::n to tMsw fl* 41' i 4 bi$* conpi LsSu:osdd*k iabatotlàSaks S amplicuns. or uttgtan*kbek4 atnpb cons. liz ac instances, a datable Label b the labct$.cPrnokcuksidmedóctabkla t: ratedinio:the labelod-amplicca areak. iCy3uth aiN irnik;aff' led toiSe tiaA:mfl*xk AmpUtation of the fabeS-eDNA ni*cuk with Cy3 unwv*1 P0k primer can produce a tecond detectable labet to tfr firs-doxeaabb labete4-nt&etule Per oxa4npIe The methGds s1dØ4kba.
In ólS itSiS:4'$ñj t*bøkPNL4 pk!=A:m*@ 4gM.Tt!e}hore$ce,*rader cabea SsoSonitALthstrunS; ($273) ft: ifls:tlt:d Sbthtva *:$dajJy,M;4it&fistM&cw jj _;t 1ib:: ttabmthtmedjtransmftte4 ckctronica ly. EoSi6n anMit tratiáidtho tth 10W4 4tcbsctbSn are uttho4, Srand *tstS* 1k tochaükaly ang *theUk? drnte&ç tSutetkal mSosa*ntati*g a. 1rtotnpdthga tm kculu wIth a plwaluty pfohgofiucjepudt ug and rapdontly attach tug one or more A*gwSiàó4wgs fimtpi:oiioikfltht*g*t*onttt rnottntlcu1&n : oSg twp: onnoiz 4intuniqiw ideØ$érregjoa t$7$ Inome aces, Sn dkks.and earn comptisc eoneuuathns csiIiS ror nIL tLt& 4::è*N:SisS 3i a MternatSiy,tcottttnMt: intht hM:s!k:: $: *flstu: *w nud tg:inthpyafoftsc1S4e}rngs. T&rconthafthflast cent etWsnucteotide tag in the pkindky of oRgottucteoüde tags is at least a&wt 01, OZ O3 4S5&6, 90*1 IISS$toatèrtIn otiaonteStkle tag in the phztaflty o(oIt,iuc1eot4e tp. Liz sotw bstam*ç the wnqesSkinotai a4:Qncbgowlwt*4agin 8tntcIcuL4esa thni il*WUctkLoiat tbtoiflwtót tjiu Spk6f *1:aeTh:S***i4tu* øsoioiipxsss of ohgonuthcMie tags is at kast about &t, 0M20, 0 4,tst6,fl.tt8,tt 1, L5,2,2J, 3 fl4,4i, 5,4 7,t 9, tO i&2 2$. *3$ 44,45, 3O 60,'70, 80, 90, or 100 thncs less thantItconccnnSni*ftat1eaone:otheroomtóieoM:Iiafl;thc?phzra1ityof 41igntMctasront:Msulccs gkst:tt:j ii% 20%. 25%, W4 40%. 50%. 60%, 747%, 80°4 95%, 91%, or 100% of tht diffrent 9ligpflCkobth tags 1n. the ilundity &otigorawksott8 tags a pr&cent 1n. tht phn[ky of Z%,j%,5% J Q%. :* :Z%4%; a%, 4Ø w;, Q*,7o%, :o;r:i%:ot diksioøgctsSkàgs kthkS. . óøpt3⁄41 tftpt a 0 w kot* tags in i: s*s.
IOftfl6J The oligonuckotidt tags can iirther cetnpt4se a target.çecifie regon urnversat pruner bn4ing sit; or any combiàst Pen theteof lit SOxtC itst*ntcs, Ut unique Idetftlfltx i4te, tbà: :óiiaoO&nagnaibe a io&ntákcnS:6 mS*herct!!z s *:pn: pn tiocyzg' ie*a: :nxtlàet*k. fl*ah koudeiag,, tt: Lãtr lèculiz 3ntotitu nkcuIe ai:S,s, itewithithnnsiuk orSy ct nthtdt* abS4ids *w:ø :: t* 1002t:t fltiS1Stfk11LtynuiSidc*ipSRNA w:4:taisw uiokcukaflinoletule a tyfrJet A*trnstisety, tkmolecutc 6I Ii 781, 4vglarxg:th 11t I,.', *b,' óàjW*O:Y tatflbetinttnucktcac4nittthzna sat' 1t*y?afl oft n"th"dsdSlos" #:,sjtw*'atiici* a:tJdM,,,wth &:14k':3"1e' Sucira *yoiSiamntisn.% wkero .tctt The' bS'1'S' :zM1tvüko?nt' can,ktSS4t"Ø&tt LOflt$fl&UflW di onlwtcani,ei*sed to 44ingu1shi4inti4w neariy sdjt:i taia p$flf,: S"e1uth":aeUgtttb'S'Skk. " ,I:i,",:"npØncjii E* i** t:4d'ói&is of *Sfr ip&atSi#uiJSksàiinS"tm kest:taupiatà pSttnt ruolect Stochastic tabtg attic nuek* acid twlecuts prior anpitfication can w$,"tlotmp'Rfi$kin ofheiwcM,":' aekL rno'kSi, whercin:uw ampUtkztibn:of nueie. Ski u1c t.Wtnáth and flu' to i1sec The 41&Aki t", )tIH &iPS*4ø,. ,!,*frTh* to áflthinS IOwls of i*t sd, .a ns1suk$ srnpJe'Thitet4sbe: a $ihe4i*d, $ac,,,','.':I$,:Ji1 :ottitc nt&keó1d kM s a ".:j':(. sW' tnoiccnks.
I93°i In some insuuas the 1betSiiudek teth molccuks m the sample are atnph&d 4 k1st4:'it:LZ S,,4,.4, z s4;ø. IGt 14hh'**' thiltjoféjcid ndècuiàathe'samØe;areamlffiS jfl:j3I i4,:t4i2o,&* 3'44S; so$e 8O9, I, tft',', 1002811.
arnptingoftbe nuc}&ne acid nttecuIn:itp&whbut:dopktdbaonheotifl'i1.
*t:!=t!,'f,',',z':", M',,'::,li:" o141$I 4Sn'ø'k:*fls *i*," S'i, snd*ctin":it Ut r'4fl'*flrerneflts SAw perS''sn'thc'1')i'$bns pro4uced hzn tbc an1ificaton or repeated ampliuletton rtactions conducted on t 1abekd-nuc3ec'a,aiotie*& epei, satnp1ingof" : ruc c acid m'ih' sam$ van otppdse rneesuzenivnS Lbr dSSt,g awthwquanitying tnpcISà. acid inolóSe.
j csa t SsanipS si o MdIUdS t pè*iàr*t nitbE: 4i*4et$$ : 11étø1$ :t$$M& tOOSI Tnont Sboditnents rnSb4ikit.i 5$ :tdtiss@n*., 4&ibn póiiki FSawnxagenesefluita A shtrnai::oftknMo itpiàtei it Pflt tinediod *M $Sg: least ste t*ijt *.:.j::aa, b):$ óiJ 4SsSWw ptdSa rgtitlSk EtS *t4Pi4S 44'ffi s *r the aUooEsktw qr zsno$ecu3e. Ffl fl di a:rnpviasffic qtiaoUftoaS óU*Øn4S Quai&atk of the óSi44iitB t:jtü, t:pnesatestnpflftS and kit acipUSns an. hybsid&ed to aatm. The:Ssthtttugtof genes Aand Il cat be detecte4 W fkorescenct and lit mtensity (e4 brbine*) of the signa an uSto:deteznün that:the qssthty of geneS is grSeSa the qtian uty of gne: A4*t4tgbai atheatixsmetiM,d:àsâkvsc ieritncInbMiSed wistar:aSt*lutc 4nantifYatioa of' genes A and B The absolute quantification method can conri&e a) siizgtiwo fl*n g*etwitItit pk1dty Eàli g1éScrSgstpt4t*oe t stpthaMicafly labeled ftiqlecu)t. w4etein the plurahty Moligonutleotxde tags wthprb3es two t k(4ffqt SjuitsrSØj)*:icyjngIi* sthasS&ty it$iUtSà.
dUtirent wuque idain tier ngknis assoidated with each scMsticaRy labeled amphca t7B dttccting the unktte ideatthcr regions eonkpthes hybñdizzng tb stothasttcslty labeled ti$iqàis:Sa iLi4:$üjt tiE, :v) tcdciocas:on:(Mfsolidflpot:szdibe nutnkrSifrcrS:wiiqneMentffierregSs tDO2sJ, If tL 1 4$bsatmaS ohs lsquSifiSionrnekksl otànear:mom RIM *tltguk Anho*'u itStçitfFIG t9 ciAtitthSè dat *!Jèkli! c 1 iisa:.4ig*pUgi4t 4 jö fe4e O i1 tjfii:n$2O)oT4j: tail 4% nR4moietth:(1914 fl: aS i*j 90 a sgtd 4 primer bbzding site (IS The unique identifia' reg 14O) may comprise a Sese. Am g. (*tIvain a tStasten& t*uIi; tuid iTiS4g (1f4*):*Mhø, 4dt:**1 fl'aflh twt in 2tht&ANA ktxe Q 960) c hfiedby SCR othpMMfl $ti4 rb$:(W9c) ádtrtt*ptfloEtg uthpd(t7fl)Lopm4un8 arnrciabde4 a4piko1s, ainpioons stqae1ffirrcg4 Th Stwsd rsQ44 cattowr a! difteret* unique idStbr tegkü& too2$ rn): 4e *n bcrrncftiodt quantij4ng gnçç rnotnmIee, Th Etho&tt9 M. s:; :* kSei ui4$urlit9 ft& LZ cOhn$iij twó**Iá4*CQO)I coflqfifl táet:sptti& ttØvti (205th. a e:idi 1ni4060) aS w niveitl primer binding sttc (2070) to ptoduce one ornat stochasdoafly labetc4 rDNA copws amèaq The mShotmay nhør thNAtóft sfc:$1s tS, st*cb*stkefly labeled amp&ons Ampli$png may tonprxse POt and TI amphfiwta the stpthasticafly labeled amplitons may comprise the thilquc 4detittflor r*oa The method may :j eáñS:4tdi t Wth4: cP$A:$*MOflZ:UJyMbd ku; PtttSn: the SSSflSttnOletS:tn c 0 hr S.flftUY*fr4 m*ü n S tt di'I4' :1 *$**ixóf 4Sina labels threadtre.tinta, . HybrMidS my otihe IRNA stqueno lt%st likely a scxnent on The 3'exon 4(1 he gene, end the uniqtg kici*lflcr ftgnoii7 SiJl,ttfStte sanwk The saiQernaycompdth 2öM &flrentniEtAscquences the màkd may mr:i:*P'S $*rality otohgmnidkoS: tMnce&1 a s 4? the oLipnu&tidbt'tSy:L* applied. a kit e nratlon o.
ixtpreOjd4ffii* 4M6:hjtmea$tei*St 002St hJtt2 00th0t fl3ttl*(L:0tab luht qitalifRNkSIuksMr a FIQ 25 the method comprises (a) ethxdw*ng a reverse trausotIptbzx rcactnn wttb an oligonucWSe ta j(2$ØQto pmduce astechastk*Iib4ëdäflNA rneik(5fl,
S
whn:theS4bdScrnuicItuk. cornprS:acDNcopyufamtNA i:cS a siiqu.oiS itS 2S a1thvdrai$tabiAth'dgf4IW tsi* &i4(b)4S': $k.:U Tho:os::iw4ej aadntbrThetrv Say:;:n: at::. S:a9k** iMZ4) prf met binding s;tø (2550) The method may 11*rther coniprat absuS4' 4uantifymg th otditrciu un MSiftrrqij tS;aftasaoSS ibtaS kyt t)NA StSth.
The mothod may ftrtber comprise amplifying the stocbasdcafly bthti d)NA molecule prkr t widdót%ting oy d:Qfl RS;. sii aSe& freteirnSS SA isa 4:i::aer: ttss::::. :ne.zi. a:jiu tflGStgcsrnc DM:tliOj eanb:ad todntzs ThWAtsgmat(2t3B Prspnentat*in ofThe genornic DNA may occur W any medzoâ knowu in the itt For exanpk, frag nay mechanical she&rg AfteruaS1y *ngmentaS :rnay ttmpthe 4tiaA.:ofsho:gØnnrnit t*4A with otwetawe:testh w1 As sbown iii Step 2o1F1G. 21. the TiNA fragments (212) cmi besfochaatnfly labtdwflt pkraMy u(âginuvkou& tags (2140) to prvdute a stochaaalty Labeled nthuele (2n0y The ehgunuckot4e tag. (2 140) mayompdse an ad*pt sequence (21$&) and a unique
I
idiSsg*i4to the 1Afrt&Th adjthtst4ucto (ISfl):møy theDNASthS. tib t:bS Iaht:SIu1e (2fl ny.oaor oSs1deiagsQ1Shemeihod.
may fith(x eompzctsss amptdb*g thntocbastkafly Iab*d tnolcuk (2170) to produce snt & hiti6éfle:fakjd i liSt. mi ith iD4 Aragriort arnpifficatiomlmxvtg fragmentsny mr:i:*dts*aflwDw ft.:::s::riu Piw vicaoiecStbe nctceitain tgniüs tho:rnethd may thrtj, tSfrbt4k*agtna& iWflI flitS. jfti:j j4: ictjà RNA meiecuk&. The RMm*izuks a mailfl4 rnoecule; Th:small aNA: nK3lkctile may bC a IfliClO,. RNA. FIG 22 depicts a gew.r&. rneU.txI ibr analyzing a sinai.! RNIA thokthk.. As s*htvai iii Step J ofFiCi. 22, cite. or rëOre niiR.A mokcui (2210) are sochvt K aII Eaca*ed V th a iu a 1i t a1&)mRieondc t? )j J he &4wont dCOtAdL Ec Q, 223fl rna eomp',c an adeptr eqt ence (2»=40) rnd a uuque der Mn rcgIon u::50') 1 he ctd4rtr seqvene 224O ma cebc a tUmenr ofthe oiouucic dz ag (2210) tO.UC rnRNA..mokeuie (22.2() to produce a 3 -stod.iastia..(1yHabe1ed riRNA i226O).
&" \bc*'k U fl Sh'p * & i1t 3 neihoi tr urSei o nn u \to'a\{u a1 Fcj abcJ1ng -kh UIILaRV Lily. lid nijcr,R\A 22f'Q) k all t ccond phirdlity ol ohpor iii it ctidc tar, 22 0) Th ccond pin ilir ofohzonuclcc dc tacst227O1 ma' Lotprthean adapter sOqtthncC 220) and a unique identifier tethon (2280). The. adapter secence (2Z90) n enable. attachment of the.vftttonuclvotide. tag. (2.27(fl Ic. the 3'stoe.hasticaily ht.bcied mRNA rnoiecuie (2260) to produce a 5' and. 3stodhasticaI1y labeled iniRA (2295). Tht uiothcd may further tcninr:re\;erse transuthuia the stotha.sticul ly. iábd.ied.miRNA,, a.mplitS4o: th.e o n a. I1 labeli d rn \ dcr.«= tine ftc to., has' it 1. LibeL d niiR N A, iu tel fvnq tb.' indC\A by aetecttv th. stochatcath laheki rnRNA i}lmdwrl the sic'ehastically ahekt nuR\ A to ai at.' at a combination hereof I he ana\ mat se a dtpul an av I ic auRNA tnt) t.tttu1e.maycotnp.nsc any cñ.he i.riiP'A se.qucnccs for exarnpIk the raIRNA mokcuk may llomprisd a sequence oçd in niiRi3asia: 18 htr./' vr,n idhachri. which \VflS rckased.Nov..201 I and lists 1921 unique mature human rniFtNAs. An.arn.y of 2 tmlhon.
featni e> car aiequrei detect 1000 aV:k hated to the 192 miRN \ jO02$9 T a. rns, thoch. k.k an scetcm' dN& los J ll. it in an be uso I for j diag.nosii. }or examp Ic, the.mcthod, kits and synes. disclosed. herem can be used thr single 1, efl p'e'imp1ant itlon g:n:tn. diaguosts (POD) Pnma t. htllengcs ub singic'ccI gene a ONA..a.mplificatio.r.asays can be. trout allele dropout anti replication bias. As ahown in the comput.anon nodeltn anai)sl\ depa.red ci HO 23A si'ete u'" mokeule has a 08 pmbabditvof replication. molecules of:1 initial copy ratts can. easily he distorted [ci 1:10 or greater just after a. fEw rep.Reation cycles. However, tvhe. labels are first attached, prior to arip'htkation c&urting labels to determine copy iiunber is rmaffec.Wd by chi.cati.on bias, so Iont as replication. occurs.. .Aneup.loidy detcn.ninat.iov. and kire reoio'vsoid.eI.etion or aniphfica.tion can bt islv and:ac.cirrateiy dewrniine.d by the.. stochus ii:: 1abding. n:iethod.
disc.Ieed herein. F 10. 228 depicts a cheniade of the gthera.l method, M shown: ill Step, of HO 23H. the Tm. hod i ia con'pn Iragmufflrg a p.roniic DNA gl1\1 \ 2U0) to produc one cit tote iragmcrecd niolccule\ (2C{h I tagmctaton of he gDNk (210) ma eompii'e any method known in the. art. For example. fragemeutation may cc.mprise conduet!.ng S rsñotkn dIscS reacUthL As ihowain Step err G<21B4t *amenie4 DAfS2O)R*o alitlab Si tt Skottac fls{2S): tt tS*UoC *:s**si i*aia 41 4(Zi% i: s:iUW *::n.its$c msy oinpthe one or mureolijnuckoUde tap (13*0). The oliwnuekotkle tags C239G) may c4r*'4 ui $fra:'tcT (3) ñ1 4ifrtWk$ü. S(2B4 ibe sSiylóoiid tuk:(ao n bearnplffSus&g oncor:nre .Sym1ti)fll43 Øfl; 0* Sbe&tSSbyLtted dthtior (23 Ill: sSiu tally lakiS molecule (2330) can bybnd&.e to a petsbe (2390)011 the Genechip detetsor (Z395) tSJ tiw n tkk a4 sys4àaa 4IMS bS' an b uS tbentrnay *t In ddnktâ&th aawuo:S Bci4 fragmtnts w hsplunfttyMelujonucteow$e tags ianpSn awuqae kbSflc* region to prothscs one or more totts*iUy labeled imtccabs and (t) detecting the stoclasdafly labcMnioiecuio&by coun tgt e.munbcretwaiq*ze;ttti bitt The isthodny tukrt1icy on sbcht'4kally labeled inoket.
WS9:tI: flcL 24 dt.agenraEsgbeni4itt ustng4intuelws&lgtiisg at tS::t:M$ ofcituWmn:L*4A Srtsybe a:Ht cwtbetd:]O%oftbctotaiflNAmtbcnwtcrrn$pi*sntasani$&The 4aAkt uz 90.24, may comprise fraguxentmg tbe DNA mc>Iecules (2410). Fraguwatafton way :tomprtwilwu* of a 4.bas nst$*m*nza Sterfl:4tagrneuSt$A moieS.trnay be stoehastically labeled wtth.a plunUityøfollgGnutkofide tttgs(2420) Stothastic labelktg IigM':àóàti M iligdih tnSdOrV: ataa produce one or asSiáSlylà&dmoiSiest thasth4beiS inokálès sr:'ø.flc.. 4r*:Ujibçled azuplicona The s4tastiál3tMaanplkon ma dSonan(24&. 1.
a t*ybotnØi5 mflthnteatt iisstii *SsStthSgaornyai) J*%$ 11*d*iài it me *gøttid tuybesyrnlSSasts4hedt: MSSsfrscrøn4ng ictoriai chemical Ubaris, Stephen P & Fodor Ct a). US Patent numbec 5541061, issued 1130/1996.
t$921: Ljbt*asàhemijófrsiucbastth kkti&,g Qtoneortnøtctwhtttdcs with
C
6:tO3 * r$peAdU:ieQ4' na Ø44*2OHA4i*;4Rb* (NA ntttitl (O). whsindtcaWA copy we&ti1 polft*aiFtida olg4 S.k; aM $b ka1tiabóJhithi eDtA t j2*2O)*itb adbUkti4fg Tnpóñng:tuthpdrncóUigM$$3*),qniqi* i1ffin(*4)a4 aodueiç2&O' (26d,11 i tp tbcS tPNA mkS! wkS b* cit ots cfl *t kálnte s&[1y kbclS.
with an ohfltztteotidk tag. lbs n*thod may MIw cobiprMe treatul$ the,qmple with wad! b:Sos,, (UDktcn, thtc4igq6U primevØ2Q)wd the:oiiSeqi&ts Ptflk$: DSk1et j$293j HG 27 depicts a schematic 11w analyzrng one or more n*ttuks The method (i* j jjng.ajjef*d: kanffiagba(t4ø anita universal pamcrb}udwg site 73O) to pcoduce a eDNA copy (276(k) of the u*NA moJccute 9dleran the ebt4A copy (2%O) conp'sthc unique MenUfist rion (21$) and the v*nsLpdSr (2194n M4i*4wkS (2*Ii . ptttstothsSy i S arnI kon&. m*ay to ts* Ills it *tJOrjSi! :tmprtwcon4ucti an sision ?Ct:reactnen:ihesSSie4fr tube ES ($194. Th: 4Skt$ktO S li yr4on Sntpu4i tathng SU: f\tbØjl*tu&t. a4**41n: F ZLih rnSod rnty ccmpsi*ntvorsettscribing sn:AtNA S:k,cuk t pSuceathA: copy., I*flä!**4t$44SP,4 SW"P,I P!4P a4 Th 11.4 :mtho4ay compth:R'':4ijssctthe ftfr4j, flflfll4 The eon stoStkt'1aSpg' t a $ ti"tbt p4t$' ott*S t*nra' Mt4:ir' 1⁄44tStt'.? beahaiphiJhc:' Pd$. i" Mtç lAit rifr"1 a4titkut emvo nitIen4e, tazfl pad g1toftycornbinattnlhereot The k,oppoxthm dthe tali in oUgcnsuckptido tag may. eowpS satpduzrbbxditg tan nt na onuck*tdà tagsybeiingWtkt:ts ôtübiàtuikttm t 4wtió Tb: S: 1.. ::$. I J 0: tSä1flbc*d dThA nxdecuki thtnotntntóthathiiafty4ábàd : nSbt4 nsy Thnhcr t. rod SSiy aicos ihiki*Iop iaekaetopnnftsceadIgcsSs1⁄4ShjtkallykkISàmpHcwttbennhqd may ris*ljglS%neor morttptim4n Sb d odstoSic$iy 1beS snc4iiwlrnt tk daythcnpScseqcjdngthe pdn.:S4aHy: k*LedaU,tTdEnaytbt ehun StiSLtTheLid ThOitØftktCeM)al. itkt SgdstSSUy1th.
aolccuks tin a ieaction baked on homo1yina tails This method may zeta GY prevnt seq ögeaTbcoligoacI$t4kSg.n3aycompriA35tiia Th$tj:Øt P naS 447 zon-specflc amp lificat o'n.
öOS9 flO12flqts.a*earamflflàien::nMt:meth*dnyamprerse nS*Iøs:wfl. & 01 u$ityr. 1iflnSotS14fl:4opt4witopt t:nAnktikS:tSt 40 StipStSoItuuhiccGtitt Tis oligouucteotute tag may contpdsc wtMtsat pnmerbtnding stte, unique idoutUIerregion and afltseqa*nct. theafttdaytihe, th4A thokt*de: Md4t&lkntak*dtycdrnpi' lth* an*iffiSkni tSJi4stkifl taio.ztie*:ai:unt4iOii:ejraifyig tht:gøehttlcMt Labeled cbMkSeSe%T? g pottnenidàg cnzyne:àaS 4Wsct*:t sThSis et RI SRI k N).TkdxtetbOd m4 MI cit sutan stqaing;pdmø tht:sEoehSSly: abádimtptThe nàIS, may tninp1Uing thestoóbsSly labited t1eStoro4usasotnn* $o$iasticøly Mb*led 4U1pfl00$, TM npthGd say fistbet compuse qnen*bg th stoàS'iflbtd*rnpconttmwdirtay*p&inbwbtS nthiitiai fl* : :tb:i,t4s0 4I:ss ThLsntbod may: seduce orprevent agts eneratedb PCL t$Si: it. dcpIbt*an cth4eflS koftykbebngoi* pv wore;awicthks by :rnnatthg TltettS ESy. c 4 *e &bhsa frtst4 h f oh *&i4S fo:: &*ø :flis*4 DtiA tIeThe:thr Sam Ii thSMsthiiy1t&kd thbNA oostj 1'rGLiii a:tcttamøbod*ts inlty iigoncor flkifl t1eDttb at&ntØtins The OtflRsCfl1fltNA aolxnkt to R*..:s::w.:y::iso aaStikllyiibeIinkce *r ihotc rnoltvulos with a pàIky:oinakSieiag# whcnrn tt*t*gonuc1ectide sag omprsCs an ohgodu eequence, swttque identi.fter seIuence 4.unkcS;pdnw;sqwo. Th cligoncSidiw ma fl pmpris: aSi:kn thzymttccqtftsat.The th niaytborw ltn'*t *ftba2qMa :teaàiznvitb * SC d Skt iSSMSw ThstUt4Jd MgonudSti4efas may comprise nsnivetsal primer bindtng shc a rt*thn nzymc iwcogtiitmn site an4s :rs*tlove ó1igonueot:' tags t*tt4tisiflgOttottMfl t*atils. tM nttbnd may Awthe compne asnphfytgtbe stochstsalty labøkd n1cou1e& 1 he method may further cwnpdsc dJg;onerora a4apttttkik mft*Si:fedxSs.fleohonudeothk O;t.np sWØ4. O,7t1Q kkC.t4fl in tfbt ytotnp4 t$ sal Wi3 $ a'*: *MthWQ) nc1ti4s ew-Awe:c edto Howtvtt insteatofTdT tailins to generate the second PCR. ptimtng site an ohgcmielcotsdt tSfiijj q 44ótiog:IM téiâ M&* ooz$j jtL4fltn chen c4 mottrnd:*rabSlute S''n;o4iotmon sktt d *n:a&tsatsM S& in F43 $ (43 Ui tgøutormoreDNA e4*3lflMA rnquI4rntöJ(434Q):cr :bifltfl.f sSwpmto I':Si(4S i:sø:øetiA a *ti I(4L AiW**Ieiø4fi*itP*< tat Th qáijjy4tope:tw NA4onesflioy he dc sd by stoebs'nic4Iy Wtg dniRNA ntkcuks with a pknbty ofobgonucteotide tags (439W Theoli*tagaycompflsea iirg spcificrgien Qt*1),untquetmifia regiOn: (070)aJ a:univasal!=netbShkzg sike (4380).
I299[ nsoM1itht thethrtkSaDNAiitk AfttsatSy t tS* triijth* i: i*as4s.ffl,,$4: 4i*4 srjms: on:nvtt #SA rno'k. the. Wb1e4 c4P:i:.4:, Tb: :ã 4 i4zü$$$ nirønpcieSides.arna d1botuct.Tbc one onte n eoflès:ot bnsyntht*tt*usoteoi tkMSM gne4pSStc,.oan: cát*4i a::::a ø:*i*t,:44s,Mj,jfl*k.c,tbt,i44te4 th:,,*S..c4*,i.*i.
At 1St lit 10 mzgSii4ècrnai*ty bet msp4dfiboi conpSatteaâ tut 12 nacteotidcs, The Labeled gene.speciflc oljgoi cat compre at least about I iwocides, Th adgcne-spcdic.ubgo campsnt S:i.hxitldcs Th dFlic àflj A:OUptt at fSd b imt oti& hi ttbft,Stsnoe the :Labe1e4:gen6tspet&oigornpxrnentkasLtutfl3$4ø; 4$ Q,$÷ ,,:,6t7Q; 7..
*fl* øsr 10300! The Labeled eue'-specafic oligo can. con!prise a iar%et s$ctfk regia The target specific regS f,the labeic4ge pecIftcahgo can bth:pSywnntiry ta at least p rtiin thtttergetm4 kct ib saint instantesç thtargtnpeeiM _j At 1S4:* M*i&tt tthat amp lent atary. flast $*f *.tn rnolectd*. AiWnstivety, the target pSflc regkn cotnpviaes at Least about 14 nucleutiths that Sc**MS. teat inn pon c.j: targt n E imba t$ S** is s iz:tfl* ideA StM 4à', ISaty,,,: 4 spetngon ando at least tut 15 lcmcn t$*j4aat a pa:d,4ôfl!WW,**ü1tThe pecffic ttgion eat iesst tnt 17nuo destrarenipbmesry p It:!cj.a pnionef the targct nvlccale the uwgct specti& regbn can comprise at least about 20 :niàOt4&tht,:átl cóio tfljfl ffl f Icéli. flo:f4t specific rcscn can comprise at least about 25 30,35.40,, 45,50; 5560<65. 704 75, i0. R5 90:911 or I O0 tdes *tm: t*tsin a$s.*omon: ol* tàc4* Ux> Jew e.
Th::tpeci&:wgjonan cotnçwtie awqueticett a Iøa* lop 4 *s;a* t 4nbS IQ p:$jep * ,àt*i:4 i*ie*..t Sat iSt:ábS% :inplem,I1nt atlt $,ISn tPOflknoItke t gtht$*.WtEfl: ét it'ean cnrsea:seqMencebt kM is Zfl4 $óentaryt at Iastapetho seEthe *Uqetnskcth. a t*nan wmpSc tsquene tt isatban about * thoatatytoathata ponlowof tho: t t1Silàt1* r4ü St':i*eSt 1:* *. is a, 4$3ø a cnhaittSibS 9S kS o jtfli*ôtth b t1Thëjjt:ót rdkn, tonçdse aseqrnuict iat iest*P/o,1erntirartoaticatcapota:ptthe tttgØt *oieu1e toosti a: s-s aag* t44% {*03*Zj The *11d *ippott cati be any ioød supp4tt dmcbsed betek in some instances the suppop ha øStc,r nflqdàqt, any;cancump4iea phws t%ir4a TM target zske en S id wnouttnOtc j 100S03j the nvshOd can Mber nipUfnn the tm'get n,Iecule pitr to hyMdivatoitto the $d suppon The ntthods dbosc4 bea an further oowpr.se seçaötgthetarg8 nxkSahyMidzedto ti: slitsupp I&anbcat1 pntSpSiS9kanjEtSSAsthardnot sniitje [003ff I theaetho& can tonriátSi W*leMaãnxtt MSt to 40 te kt'kt4atg n$th4tt $y*K 4ctst S$iS is::a4iia iisàmt St Ss.
conwriAts &iC it4kt ltbtL D (acting bcted4agct t lttUIe tOmptIsca fluomrneter Akematwclç detecting the labeled-target ntcuk vompriscs a hnmnouwtcr. in :i*er hsi detSthig the tsb*44Arg4rnokttule eonipries ã$á ret iSo:: Futther 4 Ssedls' tk s: For taj jj tbbj:ipb$ith:ott'ticäóLoènG $$hàIS4*eiMtJón* 4 Glly,tthod:ptist a rnochastkiU> labeL one or nnrenudcie:acid skdt:pth:4tbastkuUylatSeaaAd$ .:.:ø :nw SSS1c4! S uIproMeapSed mokat wi the capña4 [$301: Cpwjj ó4i*SijtWISWMóSvaó 4o*4 nøitifie dgo& thegenespt*tv*)1goscn aiáa4#.oya pe.tdhasiic* Iab*4 *1$*$fr$i$*:$$h*S S:frS5P!!*4$1S4 her$t;hgtter con iSWingIthe ohgclithsmofrcuk tarnthesapipfr. The: gtflCt a, [t1ertag The rotagaLnbMwiatbn4,rttieoligo (00*1) :A*M; 4$ : 1ø: ts$k t4p, cS,:*i* tfltStt M1* 111t1y labekd ku1c* Si a thlM4pDt. IS -isae $to iLall S *W te:W*iStn* t'ii::htks soM.u rL &thbycptdagihg ask* i4ttrndtecWt A&rtSIvdI)SS tsiottthtth*:tkeiaret rrijkculc S capturIng the tochestIatI1y Iabtled ntoleeuh comprises c& letting any wihound jiehir4 Sflylthmoi:tihat:ir:t hybetdiS Is4ks$s4 bJ4 sotids ppott4s'an: the oMsuppS4a bc'a& t4wo wriwgftetkboa4. b ithóitstã: cafltut*StdthStiSLy1ed tiI. wa**nft:*c tbt:nttbod can t eon *:n3P16tttS:the Uáfly lIbólàt; molecule and/or captured xmtccide AmplificatIon of' itt stochasticalty labeled moiocutc anSumLeculean conipüseanyoI the amp $at$nnttLSs4LsciosedMebtb tpijjata tni: theatho4 4SScihetthcan Jb.the, comp*e. sequendi. Se uSJSc th ptS4 tidISOymj1frøà StspjpS, t044t urtr dL sâ j'S hifr $4:* (: stechasttcafl, ibe1tg;aa kicMmokcuJet1,aftral%yofoOgonSa*4t:tato produce a stotha$ticslly labttlntuel& acid ntbcu1c; and (bfr detecting andlot quaitt4'ing tbLskksit la$1ó44iu41* acid it*1SIe; Th:ñü iitidd*lctik àiil, &DNk $*:ts oz14 ASnS.cthenlèaid a!cIt can, bo.dcrivttomatSjtsthJct Th.tr4 [*1st: Th,:',S,P4i* 11" thW' c4*wtsn'm tS:*4*,,**fly ta'k*uiS ac*mISlnpw!t1kIcdnsS add rn1eukaneisth stoebs*k*tUy lab*d-nuctcio acid t*kct or*ty produute, tlzcreof(tg *chasbc4y-beIidnuccc:dnempticnycan,epatedly amplMt t$U21 inome *uceiwthcn Mluftnton ctattt%chfr4ozLcer woretesbb OtAt:4è$rii$e W 1 t:hi, t*S. ká1b$ISAu14é pmd:AiteeM, aika tSdCaMt iabds ftafrd tote *.all**:4*ptiS*** Tkd 4S*ib*14t tatb* bIth,. .i tt MH:fhimtscem4y& fl tiSs idc can dyec*nbo Cyt tOO3:3i **Thi jñthi itdnttSuksotaztduø'trSiotsotthut Thitsupportcanka s4sppø $n*** tiw Skdcau fhqnSc:ifingt qingstw4q detae tItsw o?ttoast: a:oSststkll1iSku k ffi6t1t$ SiLtyTheIàkwSii add:SeSiá p:tJr Fbrear at Least a. pot oithe tndqu idcntffier rconetthc otigomtctcotidc tag *s 4cquencf4 in *saquønci4. A thtiyor aStbnaü, at Sst a pSon. tM:.SIearnoIøcuioMe swchastkally labeled-nyc Wit acid molevule is sequenced.
ti1: ettiá a*id/o:i quS}kthri of the s p InI ly tábóiktucl*act& L4%PS uz4otthe b4 ilL m4e ii*kct4q arnjiLiS& tàW pantffiâation of flit Scbatic ly Inud S:Thctd&c:totnpsc 44 t*ièzi or at abi 1 àttà]ád @Sfr44 nucleic aS motcvtztes a' products thatof can comprise aay of the tt4ctk>n and/or Pia 1p Ama$oe::ca ii4S ttevt aS:qanifri Sly ctoMmckkae:imlecdies or prnductnimrac 4y$dt 6be o::Ito detct iabet4c1*se4otàcuksocprn&st thaet tSitsj M áSSstSei sl&s item t dIStdsteciin aSS (4 iodbastiS' flnghrreth1 rnøiSleata pSflto1ólignuókótkJtags to produce a Mocbasticatly lebckd-nral zwlecule and (b) detceting aad'or quantifying t)w sodSikaUyI1àbek&*aLmoka4&ia:some ipSb iht*d mqkcóla:arcnucl* aid molktcutes. Tihxuuckic acid LflOk.CUleS can bc DNJA or RNA.
{{)OSi 71 The btthod caP further caniprLe conduttin a everSe. tn± PMtiption rectidn to it.the a tochisLicaLIy4abeied eI)NA. e.y ofth stOhacticiiy-bihekd viil m.1ccuk* e ocht Ly 1abctd \1 R\ A niøkc Iz I 1i toh \uctlI>44b1cd i 1 moicci. Ic can be npeatedIy rvee.fraiivibed to prbduóe rxiftix4c.tochastica.fljH.abekd eDNA.b.o:ps of the stoch.astea!.Ev-4abeied virid molecule... The methods can thither eocnp*ise. ampli±iing fte StO a} K III) Ic! wlcd ira mulct t. Ui Jfl prod K}s thctco c g toUns Kafly$cibYkC L DNA opy o 1'mdii c roha,R a] -ab lid \ oat ann,IK on Ilk. ,tue Ii i,tnalk taUt Ld- nJ rnot.cuk L.d he i.tpcatLdl amphfiet Akert nnc', the preduct\ of the stochasucaUv a)Ucd siral molec&c ctn he teoeattddly an'phfied En sonic ntarlce' the fldIIL oE the stocha.stiea.tIy.iabelec. viral. molecules toe the:stochastically/htbeled eDNA cooies of the.
stod.iaaticall.ylabeled vira] *rnolecdc. Aheraatively,.the products ufth.c. .stocha:siiillv1abeled na] mu eeule a e t e tohateaLyabekd uat amphcon [003 8j In Cilk) iccs the Ni {hod tn the &onvu. , a( i. nn onc in noi deL c' iNc lalels. to the stochastically Iahc.L& -viral mu keulca:. or pn>ducs th.eret1 in some i.ri»=tances, at teast one detectable label is attached to the stoc[iasticai:iy labede&virai molceiles or.nroducts tho.eeotAiternatise y. at least two detectable labels arc:attachcj to the. stochasticaflv ktbekd-vira] mokeales: or prodd.cis:hereof The ddtecthhk label can be bioti.*i. .AUàr.nativi.y, the -i i" netccta.bie label is ailuorescent dyei iie Luoreseent dye can ne a. dye or a)tv the I he (y iie can L C v [003i9.1 ic. rnc,thed can turther comprise.nytridteahon ot t ioeha,sticadv iabeEetj-vwd.
mc cculc' c-ac ptoduet1⁄2 thereof to a solid uppc1 I he so d \uplert cm he a bead ALernatnely, the saud suppen is an ana' 1003201 1 e methon edu tuithei LO t. em daetmc a scua cli ri it'tiô 0 deteunni, the qtenc of kait A potion ot the \tOdTh\tkil) hIbekc-\ fldi irokci k nro(Lb thereof orn-ln'knICe at teat a porbon or thc ohgonut leotide tag ci the siochan,a l' i,abcled-viral ncdecule or pro Luet thereof is sequences. For example:, at least a pardon ofthc mquc ahmtuicr region of toe ohgonuchx'ziuc £59 is sequenced In aioiher jarre al tees 4 portion of the.tarte specific regionof the oligoi.i iickond.e ttttiS. SetliletiCc ci., Alternat:.ivel.y or addition:ally. at least a po:r)p of the viral ni&Ccti.k oithc sto.chastcaiiy labeled-viral.
moecule e4uenceC [00321 I Dc.tzctkm and/er quantification ol/the stoch:ast icall hibelc.d-vind niolecutes Can ccrtrn se dctection and ci quantification oldie ocha' ic.L\-tahcled eD?' i\ es nd ot the stchasiical.lyda.heled. viral.. amplicons. Detectioi and/or quantification of the.stocfiasnc.ally iaSJStInokctiks can thàhcrcomprisedSctbn*touoorrntes4AttISbèLs 4 atbed o s a s 4Jti*tiö$6fthi * . StkI b&4'$ M:øtØ41fl1tM thwt4: fldStat4iWtntUthdLs&SEtbS* Ponxampi&* :nilàculesorprndSwthercot Al fr4antmatnywa& cau brused 4sSasd4t tszzi iii mc tat buscdwde in4t viral load inJ:st bjeettt rinfloma. viral hicSot 4*:.tht q$4o4J øsi oS::S i 1 44 *S tthedi*wéai4oi' p a:otavkaf nfrcItn, là some qs 4iflil 4ctiàt in tiiSâMug to Mit h*tpU,fr tOOii. Fwttttat b EhS. ds,:Sg,and s*nfl,dfl4tionniS, kiS sssan1: bImarkerOóneraZly, the nSmdsS a4systemscqmpth, ®sSztió* taWflnga fit tap to dunbust4clMi*d-blo marker, and (b) detecting and/or quantifjing the stocluethealty abeied.biomarkes The bän*rbr can i a audqffiarkeztbe biomrnSca* tw &ni ntaM pfsàmnte Sè:aS jSe tan b$A.wS$u:k4erSt y,hetsk! Altcrnnivcly, Sbss: 4s'4 tram r p iiiM1s:: tOfl324JI the:nttho can ñh r: Setonduetinga:r*a produce a stothasticsily4abdct eDNA tøp otthe tothastitally4abc led biomarker (e3.
be tepeste&y reverse transcibedtopmtcc k do aly4abb ct4A Cofts S t1otaakn I: : I' , nM4yroranypvt& th ft tHI&ted øDt4A eSs pc birnnarkes ct*u be repeatedly eqip ftf led Akrpa*rel>s the pSsts ofl)e flochastkeHy-EthetI biozttaSm fl:t 41y amplffiSL Ifl S in Secs.tprndutt8 of the $SØi. kk *Sti:Sck4 IDNkt'SoftS dk&y$cledbbmatr Ahenxtiw1y, the prnduS4fthethMticaffy4a$*4 bion "r k,,rs are the stoc.h.a.stietdly4abded bits n iarker. a'tip.ii.cork..
{{3251 1..3fli ntatt. th> tneUiod fthihcr compri4es attattthig me.or inre dtectabk a1)ei$ IC! thC. :j:hbekcl-t)jomarkers o produet. thettf. efli!flStat!.t&, at btt one cieteetabe kibel is attached to thc..stoehasficafly Iahekd$iomarkc.rs or products Eherertf \ .ernstneh. dl List tsxo dceiab1e Uhe ue atL,hed to toe stoc icituijh aheied-biomarkers orprodu.rtsthercot. the detectabLe label can be botin. Mternabvely, the Octectable label, is a.fluoresceni dye. The. I ntecett de can. be a ç/' dye. cr a. TYF 56 dyp. Th d c&n'he cy.;.
[003t ?1 ic methoil fijrh to np rc br chz.'tion or flit stoePi ucaih.. thelec-hietkeis coi any broduets th&teOf to a ohd suppOrt1 The.oiid suØpoti can he a bead..
Aiternauvel. tite. so.ud support is.a.n anay 1(0327! Toe method can further comprise conducting a sequencing ravt.ion to dctenititi.e the seque'nct cif at least's jiortie.n of the. stdebasticbuiv labd1edNomarkir cd. preduht thereof.
In,OP1C insJanCe\ a lca'd i p00 an QI the ohaOntHeitct " of k,tochs{ LJ P$ htheLd-bIOJkVKel or noouct.heCfl N CUCOeN Ut Ct$u1IC at least pomoi of the unique identifierregion ot: the oogomtc'icouae tag is sequenced. in another example, at least a portiofi. cttt:he. target specific regk,n of the obgo.niicknuoa' [5i i scou.cnced.. Aitetnatieiy. ot addirio m least a portion. of the biotha±et of the.stochastlieally labeicd-hiomarkdr i5 sequenced.
1øO32I DttiOti.tmd!o.r quanti.fieati:oti.ot'th stochestically iaheLedbiotaarkers caI Compnsi detection and/or qu.ao.td5cation of the stochestieafl.y4abctlcd eDNA cop s. and/or the stochasttctllydabeled hioniark,er amplicons. .Dctection and/or quantthcano:a.ot Ic stochasticafly labeled-biotharkers cah I thher coMprise deft/:tioh of ohe oi more labels attached to the stoch.asticafly hibeied-biomarkers.or pmd.ucts thereof DettxE.i.cin and/dr UJdtl itLation ufthe sud asLci itt Lbded htomarker 01 rcdah Uiuu,fcan c.ornpue qns of the. detctio.n. and/hr quantification methods disclosed herein. For example, a fluoresctmc.e reader can heused d:etect and/or quant.b5' the stochasticaily tahelethbiomarke:rs or pmducts therecf.Attefnative.iy. a rthcroarray reader caiabe.ud: . detect and/or jf\ the swch.astical.tv l.abeled4honarkers or prrsducts.th.ereoti j00329J th acme. instances., digital. dct;eetior andibr: ditzita.l quantifit tictn ot the hi.ornarkers *ci be dsed to d.iiwnose. or progno4e a cooditioo jp a suijeet itt oced' thercof In s<,me nst.aricos digital detection and/or digital qu.antiiie&iion of the Iho'mark'ers can be ufl to Mohitcir ithe.hpcutic regimen.
{003301 The cond'itio can he a cancer. The cat cc can be's sarconta, carcinoma, teuke.jnja, I? :w IS$ijI AItthnqSitn SiiàStii.tsgS:SSS tan bea baaerfrst orvjt*I i4fecztb)m, tOOSI. ?OSr S flS stems fruutiiior 44tØuiiithi:i Cr4tñ 1àiôaid iib 1oo, A pnMdrn a plurality of oitgonuekondo tags whcrem a oligøxincleotidg tag comprises a a 4. :4 Thg it 14 t ski Qri*o:.E:n!l: ë a Isbckd uikkS tSlceule,, tsa st.sas i otattachMg to acligonuekotMe taa with a uniquec tdinLifteE seqaéntc; and (;J tteotit G) of the nutc acid molecule1 e a:poSn *tof.Sd (U the UligoktUóIcOti ta:á idi*iS.. oj.. tdx&ófltiSo$ tdMt i..
p.dflSàkrni*Th4toSors.:kS IabSttac4ShS thereby counting or dezcrmlningz nvmheromnuclé acid mntecula in tbq»= sample. Th :rnctbod.usty coninp. 4.g or deS.waul*r S&.ny 4if4:'1.4Ii4.:n:1e:1* di:ntiiudeiaiditcct4a*nij4iifWt i :a I tides base -the b.Snuci* ad4 may betousd sinmltaneo ZI*WMA*.4Ilfrie$ti4 add mo lectk: .:y bo:tuS euS4bc tos:si :iD flSC:($ pMd a wal$ otàhgcithkoiidt'tag' wherá a lko1 r:t iiRüi*tW*tëktUet a tacât s4'ñifle at$ an Qptww4 PeR!=mer sequenøei (b) combining a sample comprhhig midSt aS ntzuks with rk plurality of labeled pHmerii so Ibnn a labeled nucleic add nthxule, wht&ein the attaehntnt J$ôt**k4 st*s$n!=uf oeti*n t4eoktagjwSibp ot*dithi,1:fto.
4$enatheg...nt:ornumbtdzffcL4nrJabeicd 4t tóMSisthcnqk:j.:: ntk * rpflØptSr 4 4ojj:dfll$:uItt *tnSiod *icsk:. Th' :fli diftbze$ nuclS acida may be: unte s mu ancqu4 A*tn.thIyj 4jfQt nucleic..aeid moecu1es maybe co.w.ted seqhsn.u.aI).v {{)03341 The btthod cftourting ct doreiinItüg a aU.rnbi. ofnuck.ic aid i1Cue fri a a:itii Ulil) .ii oitis& ($.) prov T diiig a btUty of' biic:ec wherr:in the &4wont dCOtldL oriç' ic a ta. ttpcedk equercc a uniau: iJtntfler equcnLc ccnipriiq a ribent.cietç 4CULI, andan optl.on.:I}:.R. primer se:qu.ence;.(.b) conabnm& cc1Tp:sjs1nL: 1W:CCU) tICidltflOl.CCLIkSWith. the Iu:raUty* O.tcWgC1flliCkObdC tags*to tbrm a.
nuoktc: aejd rtiohtc.ulO:;: w tcin:a. tract nucleic add tno eula. is capabk of cm.aehtna to 43l1oflLLL f)Litk. Ics5" ti diII tt'nI 11 fllt. likit fik t quc flCt. ( ) ) 0 SLflP 4.uf) (if Ge htbeicd nucleic acid malec.ule w.hcrdn the copy of th*c lahekd nucleic. ecid mdkcu.ic.
ehmtth±s a.cO itthe ntc.1.cic sid tnoiecu]:c. and. copy of the OJi:t ii deoude tagalid the ubonuclete add of the uuiqu.c identitier seque eecornpcisasmpaced with a deoxvriixonucieic. acith and (d) detecting the copy of the labeled nucleic acid:moleculp, a.
cemplemctir aithe copy of the labeled udeic acid noiecue. a rcverse compleneit of the cc py.uf the hlbek.d nucleic acid.mo].ccnle., o.r$ portwn iherec f o dcenmnc a Count ttthe copy. of the labeled nucleic add molecule. thereby countin or determining a nui her of nucleic acid. molecules in the samphi. The method may eornptsecounhirlg or detern uftig a nun.thcr ofiaar more diffbrentnucidcadd inc.,leeffle The method may comprise. counting ä.ttueiher Of O or wore difibrent itucleic. acid molecules. The. diffdrcnit nucleic J( id nok td' t'ia l tl'i 1)5 1 oi able i utica dry, at ba'n pan S OC thc en bflC h'i ua maybe. canted si.mu:ltaneoitsly.. Alteiratively. the. diltent nucleic acid jy le cowited.scque.ntialtv.
1003351 The. method ofcount.ingor determining a number oIRNA molecules in a sample day comprise; (a) äthibihilng. a sathIe baniriirg RN.A moiec.uieh with. a.pldra.lilty ci ohymt.eh otide ta, %\ht'le n the uhoonLcleoutt. hi eom es an R\ k-pcc W equ nee, a LuqLt. teL nutwi sequcne anct an optioal PC R numt., cqudncc, b) mhc,izmg co of an KNA molecule by attaching anligoi.iucicotidc tag to the. KNA l.flOiCCUIC tø fbflfl a hthded D\ A moLcule 5; hetci i t.acn RNA ma lecu hi eapdNe of machi to cbgenaelcotkte 4au.
with diffrredtunique identifier sequences and each labeled. DNA mobecn.ije comprises * of tic R\ \ mtkLeule aad COP) at the oligunuekonde ao ii ci (e kiceting he tabLied DNA niolecuk, a. catnplcrnent of the labeled:CNt.A rs.tkcu.k. a nrvetse;brnpkTnnt of the hi.bcl4 DNA molecule, or a pc tiontheceof to determine a. coci tofthc lbed DNA mo iccuic, thereby cottotingor determinhig a number of RNA mcdectkS hi thc:..stxnipie {flU33 1 ne method ct cainnog or ictu mnm:ig a nuaihei aIRN & moL ic' i a ina com,irse (a ios id cc. a plraht at ohttonuelzonde tags s;ieietr. the ohgonudeottde unpri3ea ait Awspct,i seqgence a. unique 4à&ncquen cwmpdMg a txaiidéfcti4ifld afl aptional PCR. iithd!=* whesina agS:RA lAbdflWu&wherSfJbe ijjijlód bNA rnniàc hi cønçiis *tøpyoti4M etukS t iigonucLtpndThtr1bonttS aS ottb u4e I[abcSPNA tSa icntthkiPArnD1ectM *itnz otthc labeled DNA uxijecute, ot a portion dweoflG detertnine s count of the abe1ed DNA mojccule, hS, caMnthigq:4icninbganwnktofR$AnkqaàM dnrnpk.
tS:rntdofsbntgettfl:ioAbbiik lu a ossng *N&n'p 1j* tthjAziatItôf oUrndtherd' tathSj!=tkbL ouieis eapableafattacbmg to a dtSn 1abdb) o*ionally attacbiigs eeond oh teotidcia* ids a *W1Arnos:teofonad1ubektRNA rnotccukc (4 synthcEWnz LCO #rM (A asi,siee:::s &4DA lab bNAntczubeurnptiasapy ofttg ui ágSa:ypFt RN*.
molecule, and (4> detecting the tat*kd DNA molecule, t complemein of the labtldd DNA *1,sñ4wM4tp Stoctdc at kmatftbSkat4thlbd OM mokti;t'r tottkntctthhe.
duM4abaS DNA mokeuIe, or a portion thertufto count or deternune' the number of 4iffàentlábéli DNArnøieuksatbá tuiIS*t DIM m I cut s nnng:anuthet:o4A tthsarn$e 1003381 a ftotoSWi::atttaØt àiifld& KNA àdà ma. S:k may mjS g4nrnktonStht$ tWAemo1ectile wkq$or4kyófità tb I tM Ab$k4 1 4POWS *$ ØhtU): Aob*:thh' to addbcnt bptkaiy aschhasconM lU: tvrnat4abete4 A Slscuksnd(c) 4it S S*tAiSse -* *flSjabóte4nul,ceokMeióftjpWkd4N4 tSicSt itd l*bC13cj: 1U4A j4 compcent 4:*7:*flRNk$1of t::tt tKcnte thenuttóUiffrtnt labeled tA moles ovdtent dual-tabeS IUIA so nK3Ikcti.1e, tIii.rcbv coua1Lu o.r dzteruiimng. a number. of tNA n&tc*euios ki the.. .sanrpie.
1003391 The btthod <tourting ct:dreiinItüg a aU.rnbi. ofmRN.A itkcuie in a a:itii Ulil) .ii oitis& ($.) providing a btUty n F biic:ec ide wherr:in the &4wont dCOtAdL Ec Q,OPf' NC, tL tLEsXCfk equercc a uniau: IJLntfler equcnLc. arc an 9ptIiOn. FCR.. primer seqen v *; (h) cçwthth.ing Mmplc x.mpri.sin mRNA neLi.ie w.th *.hc. tags ietbrm a.abekd. iRNA moiceule. \VhCfCifl each target.
iik We ap thlc ci aU dung o i thqrcrtt o gu; uchct *r 1fl synthc ing a copy dfthc ktefrd tnRNA molecule to Inn a. abcmIed bNA molccuik wtercin. the Itafried \ \ rnoLcuile corrpwt' a cop tn (he n$k\' moLtule dP(! a uLotv Ceondc taL CL otihe okwt nci.eotid tag.; .itd (ci dtecting the. labeled DNA. Ic. a complement of th.
labeled DNA.moheuie, a Pvers.comrlmv1ut of the. hibeled DNA.mokcuie, Qr' a twon thewol to cLterr nite a kount of dt1erer Lubctcd ON k moDitiIc *hcIeJk OU1 LU" Of detornwumg. S flW)lhQt Offli:RNA tnokqffles in. the snip.J:e t003401 In one p& den 1 tc d RN \ rmn a I o ai als.n,d lr.nc -n I d:iscloscd herein.. Aftcr ecU lysis. thc.polyA. RNA.may be enriched by.capture.on a solid nppeut.tuch a u bead, ba\ ant obt,o a attaLbed o mc unphticUIiofl can be N fhrmci on the lysate. A.hJ'iek'd cONk copy oftho RN.A is made.. 1w hvb.ridiz.i;.m a prdner that has:'an oii.go dl reon and. a kib&4a. regidn. The. J:a.be1ta regihn being 5 oftheoiIgo 4!' region.
T>it. ubk th.' c o m iuph I h.at M)fl n.'4Lh't. hit 1⁄4 *1 Ii I C'l4ci I flifl' sO that tht 1ábJ ia.ti,regon. MUchIS variable. .!tqt primers,, is hetweci cothmctfl athphtIca:t100 p.fl.fl sequence and a 3' lita) rnno'n. Second strand eDNA. i then. vat:eaized iitw*:sndarl methods, ft'r example. use of RNascEI and DNA polyma.rasc.. The resultirg'dsDNA then he Ru.cad.y athpiified depdnthn.i' ad th' adipli.tieation prithet Fdf eampI.e, if the arq'hcanon pnmn uu e h 1 17 RN \ poI, mcae por loft r s qu tk C, ltlscn',e RN \ ear' N zoneratcd, IVT tinnt TI RNA r°1 lfthe smp tfkatuon rnrrc sx aenec nc ud s a site lbr s i eking emyme (e.g. Nt BspQI). nicking. cnzy.nte strand displacement can. be used to ctrierai-e DNk copies'of the RNA tsrce!& Tke:eopkis. can then, he modified to thcl'ude sequb.n.cin'g primbx.s at' ohe or both ends. and the prodtets can be sequen.cetf SequCoce information is:eolleetcd.tbr thefl cam and etw'uab. oft he adjacent sequence to.pw.vide an denuifl at'on Ott It' t %rge 441 the' oligoumekotide ta'g.cOp'rhe4 e rthonutc.k.ie acid. The olq..onL. leotide ft a ma onuu se a thmt se t. ac,d that N Ufl-s tI 1 boo hgonu\. leohir bus nus:omnr. at onucUc ac d that t cytosiue Ihe oh4enucLonde Lug ma courp e a tthonucieic' acid that ia adenine. The oi.igonucleotide tag may C:Ompt1Sc a' ribornjc'leic' acid that 8.1 ILguanosine.
oc% St:a PS rnMi& : S Spi$a *:4M$$.
tOO4%t t ffineqstctfl onuábSo tag aaybc'spcéftt fr l. *:4S[tM a*i1PS s4:to$44 tonthes mn3li cqit Th tht $p $equce4ftbe 0003fle p dt aluelict Lu s t t:tfltS.
ó;i.øS*E áti* ó$*4 S44j; fl pyefSlaIlSDh4Atk4t tAayte syntkaizSbya tta*cAripfs CJ3Zfl$ The rvers traoscnjnase enzyme n*y be sehctM fttm a pubmn; tS:nxt&n flitS bi$priso St a oop' dl fhb laStS tiIciid tiI. lS*tó rIbosiia s**:a::iis, tOO4L I MhedetettflMcp S. thecopyoU1 Lsb*d nuclek acid uxilecule. a complemn* outim copy outLw labcled nulek acid ntolecu1e a tev&secolementóftecopyoftheiabeldmJclókacitmoIecit,ortporHóftlbenof In mthc n'Siio. it I niienie molecule poru&n øtThe labeled nudelo aelt t*kt*Le, the olipnucleotide tag of the Labeled *1 io floiào* (ag 41S:, Wóti4 soiøiOt Stoctdc at. lb tth t SSdtkAgo it Sitilë>a snis: twrs1:i 1: äe:Mt* :i$,, fljj eopy of tht labeled nu*k add nitul; the oligoitucleotide tag portion ofthe. topy efth4 I*bLcd ttic1S$1 k:*compkmS thai 4:SSó ó*ttó a thereof,for;aDy onthIssanoonaua:stzi&.
147IM, t& thb &oflte. :*:4:h*'.
óLnudniemmàfthnopydthphgonpcip6tidctAg;anva eornpSettt [003481: nM t$t. gp:SI:4SS, a 4nft41ØunagJunronp1ement4ft umque uDJeeul4gjwIctbn, or a pottiop thereof to a st euppod The detection step may conSe h kidizat:oii copy otthen ueaIec*tajunctom a:cex*mentutthe, :cpyefltsawue rnekvu à[ agjwictiou, awverseeonip1èmei*oftkcopyeflhe unüiue aiSt Ie4aget1on:tap:k thtfto ILid s:p tOOa4tL bismØ *:&* 1J4A ft C üj Thi *t siy atShetS the Mit. tny.tnemkr 4OmpdSCtpwbe bth tot sch possible Labeled nuddc acid moteøuk coznbiuaton. In another Set, the soffd supped mty tonçdse a beat tSSi Thtsi n*flsd* i*ai. ófIj tabelednSd4zscsd ó*!MththT4i*j: cbnçkkncs thcztnfr i portIon thttftof and (1k) the oligonuckotile tag portion oft labeLed Auclel acid moLecule3 4 completient tbeteoi a tevetwe coxnpleinan tfrseo cit vortkiherecE bu,, a pe JhSifre4lqn step compvquaicigut()4kcntwIâä tid tthtüle tiondIrStopy ofJab:tutaSleui1acota$obat4.
4 S:: *io:. hjSoio &to: ::t:44:W) th o::flSi4 *t$tU t:opy rflt:Itheied iS k S2taittute ft**fl i*:sat*e deteSn £$p may eonrie:sequenSgbe unique: àägoaxóStMtatYNA;jhakwa tonmffic us u1isonucWbti4* tagaisA :iwø:le*t pc. *i# i:-pNAj Si4ót *tti tkrcof in some aapettç the detectiofi step may *lrqpth4 smpteadvig,tht copy of Ut unique oLigontzoICoti4e t-I)NA julwtion a cMwlenttftt of the em of the uSque oligotnwlwdde t*g-DNitjunc$4s s jkMctiobyor [001 âStJti:$Pft @;lstssa s*::* i 4'$ th copyof%e:: h led ckicaci3spence: iampWie4.1bsnØfta.tion:&t labeled nuc1*asid mokcuk es tIE copy ofthte ktbekd nuc lets act nidec ale may coinpdse a PG Lbaetuithe$. jthóPCR:4:j qPCt ThaPC*4*j nmympriseRT'PCtT&flbtwdhStnqconritemuJSAPCW:The :ttficatzonntthe:nucleic: s$4: Øla 4nJs*:r*t 4.*:mPa4.4 t&b*sed th ma rStsMèmpthni y astnMtpiaeenteat:P$$M INst tjjèr t *S*i 1$ $m* 1:M!j Akói:W tk ltik1tAintp1uSl1iyttctllatn4bt*ornic V ubout lOacet 1$t Sn&4pidnccukba tNkmoequ1ettnucleic mcilècutgc The xSt aS melecukxnay a *ewodingRNA iwIecUktbo aetcodIng ks.
t A TñnntMiifl14 mOltcuItd*>ta øaia\ tkoklts4iec%tdligcidVklde taskatssttnuckistkuLety :$i4 Th ils:ip1S% d bt*S1& lt$b14fiSiG& tS 4 Thr& is a::wt.
flóuIO*iit () a tat scquthte, and an:ot flat PUt primer aq4euce; (t) tofltbi*ingja ample oompMng DNA ntokc'uMs with ft* plalky 1Iedptirnste:Lrma1abchd PM mokca y4zeijp tMitjcjtD}MjqJccuLi cornpii's DA:4uk:and olipS Mb tath s. tD4 tnSjte ais: t: iSt $I() 4çgtj(i)t}iflNA :n1ócukackrnenSil:DtASlecnkaa nn$Stnt ofóe:tM oISt cnportiontbereof and (ti) ut oftgwwcleatlde tag4 scGmpemcnt oft oUpuuckotldflag anwronmcat4:the olinuc Sidüagorathlon btootcrminc:f count bHA:tr*ktuie tlkabyern& ng ur:dnnSIngsnu1tGt DNA ntt 1k the sample t$6i h:aapS:1 aSho4tSithth*r4àSMg.a:nwnntDNA.
utsianp:titiaig: Aj:'j a unque4cnufler 844w:: ajk a4e*è, ádt o at :Cky*gnt s eothSntnapk 1 ising b&kSS4 wiii tkptSy of labeled primers in bun a labeled DNA nóecule, wherein tbe attachment otthe DNA molccnle to the aliganuckotidé tag lbnris a unique molecule-tag,junatzon, end (e) dctecIrng th unique m tuk-tjuhction, a complement ofthe unique m4eeule-1sg junction, a cSEeàSnSàtthà iii4 Will üétion, O ajà** ttáiMiátth4Sbà.
cntS:ornurnberoMiSkbótS PAmo1dcülesJSre6ycoumS::ordâaSn$g,a 4'n:. øw sss t405ti, ampleeompuslng (a) providing aplntaiiy o$adapt*a whereM the ad4lors toinpntie a a,M$ii of to*9j*h kbth uióW compnsing DNA fragments to btnni an adan-DNA fragment conjugate, *hezem
PA
subtanaThtThøflNAtncxandwt1yattads4t anadptorwtauSqut ate SMflEftk Wpra:e fr$:*óft tte4f1øfl#t- i$W *kfti*a method:detennrnm topy nuniba' o a bqPNA :nxtkctdé inasampie c*sbg) pro%dag tphSktdrsctetthcadàpàs corn a tiq thtff1Ondqu3e antS ada$ rft t*p4tWs dattaài: to a dikTeiS:tA **tM M J1kfli:::$ ci ii wik1flMAto i4::..
Ssat cbn)nsntk DNA fragmtnts Cc) attaching adaptors to die DNA fragments, whettm iubsLaeltiaty aft of the DNA frams apattotbeingtantknnty attached to an a4sporwith 1hc *ithrne* o a4apotoThc DM*agrne$ :m* a tthA$uncüo* sdctecdhg iifl Mab14Ajsotia iót*M n i:' S.aA4*4n34**s có4àS:Atthé4té sda$br;Djuaet1on:at ottbn wSo, detóSétttwuntttnnmk*z of diffes1i unique adaptor4NAjuneta thereby 4ctaSnan a copy nunrber oft target DMA.
0*3591 In nnaaspecS a itmu acid. tnson* aspccth4t n:ais:ithic iniotnc apa the ri6onncSttacid is cySa sorne:a*DtQ.
S$* p t*k iøø **da.$ A s*he S**o IA$ 1S14: [00364J In sontc atscts, the method tkrthcr tumprhs syatkesISg i vopy of the adaptor- thootjnoi*ffla jj:1 sot upSih doécSgswp.:,, thttoy of S unique.
P: qfi*$*DNA$ndi* tt%tntGmpibMtflt4ftcOfOftheD$iqUt*1apMt.L]NAjdfl$tOfl,) QtWPOi*flthefeOttJit some aspects, thc detttmg twnptiscs dtncaing the copy efthe Maptut a complenrent iutiOn treoti 1tM tp thb 4::* DN*$'a:pIcnntfthunique tiniqtadiptor1ncUoçora$nSthorftti tsoüdstpport In antEtsspe mteuos. dj hb$ jzatjbodfl ot* Sfl ofll* topy.ottbc utinadapSGiAJuSkfl *rcversttomp1ërnSStth aioiw apon ftG363i it:sonw a twsolid supjrnt compth*&aaay. h as :tençt'kes pmbes:atacbS ibesurce; In senzcaspecS t4waay:,pS n probttaturc ta&'r:b!$xjur't 1fepir: i'MO1⁄4Ø PM $ctóà. 7AM4flfr$pLS 1id4jE**; onçrtse a::, In tkSne :iDjiifóña to: 1t:t$4 4 *TtDN;j*1:S *i tonmS p itt ptor4*th junót t,nr a:pScuthereot in s:aspectst 4ótettlwt St4 iMtseqpentng4e tS uniquo *thptorbi4Ajuàta cotn$emcnt f the copy ctthc un4ue ataptctr-tWA ptaia a. reversss ccmpkrn Qf'LIX, :oftfl4ut S-bMA jrnSr at a:pottMntheot {*0364j In soP* a$PetSt the detection Mep coiiptLses equcnsbg the copyoftbs ad*ptor5 fllllpkwfltOtthttofl ott p4itoz, a:rtse £QIvpkl1wntof fficcppy otihe a4aptor or iØOdkt.flt li tpa&üadator-tt4A Eapnst cO is asp I [%$$f: tibnornslitms tstp*tin 0,) pnn4dM plur*18y etoJ1⁄4snndcttli4t tap wbetvia ofigonuckotide tag comprmn a wikue i4cnti&r seqwncc, a target se4vtnce. and an optIonal CRInconibithtnarnpk;coinpSng #Iatd. pStsuts:gaornicD4MiIigonncIwtMettig eanjugate, *&eSatht genonuc DNA w capable of *tttacbmg to a ofzgoaucbodde tag with a unique identifier seqtace, and (e) deteetrng the gemnnic DNA-oUgonu*otide ug conjugate, a complement Mg DNMUnucode'tbnjuflk,toitonItfl*m4tth6g$on* tMA ta: g iÔtA$rtn bøS. count!= 4sóSS unjboc é dmtOY b Ao1i1thSikk abSfgiOtisJitiei: toflM6I Ins S the *Lë*lMg $ft. eoSC9teCtbgt gcnomk: DM, U: pt 4ftlfl ncDNA1a t4'ttó ltoftht<gcnotflj:tNA.ot a$fl àtS pà thi, S*fgit, 4o*flsgt*olig:4to*ido: A:E aoftheomCteottagatWwsecørflatic L$4tit t*M, Insnnw apt S gaabnontycurnpSaaancupok:The dysnayootymay1,e fdasht Th*s$** Th:s*s:4ss,Thii*;:1fl: **i::aad:;:' 4:::1s'::n aspeØs,trS1dmay:fithev diasing Thrnqr cx4:JrQi1e In SO.1 ne aspccis. ttK. method.nay fuirth.er.euimp ise di.agnosii.ig I) own yntho.nc. ILki omc.aspect, t.he:method. nav ftirthet oniØri»= diagmsin.g E.dw*rds syidrome ih some i*si*thtIs t*-e tyxttht;d. im t%i urhe c Thprise digiis ig Patai,snthiiix. In sone a4pctct. the gcnLtlc dtnsort)lajt, COfl1flT' ddetioi n the gLnonre ON A In itc a\pccb rhc neti.
ahnorrnab camp ci polnwphist In,onie aspet the p.neth. oun.Lty umnrnes a mgL gene ci soideN hi aspeet\ the KL.eUL abno.uahty comp i'e clrojnosutnc ttafl5k)cl1(n.
fl01ó8 if) .QflK. tS it,aml)Ic' 15 ftC)Lfl enil yo I O) it Is, tic' or ph.
ctthnrises at Ieast ic cell froni tht embryo.
1{P03691 ft onie uM'ec 5. tne n eti ud!\htt u nnipt ses detcn mm og ai ur p1 mlaum' of the embryo based on the detecting step.. h s.me aspects, the genomic DNA is ftagnw.rtted.
prior to attachment di the uhgonud.coude tags..
[0.0370 F In pme aspects the. gceotmc DNA is.fragmented by a restricdon tniynic. in.
SOTnC}h. tC lomb.. DNA I COO flR 111. I c iespe Itic i esP h nfl fl1 VTflC {00171j ft c'nic asreet5. the oltgnruc.conce tig COIt.prhcs a ihonu:tcc acoi In crite tie mionuc ic acU n macil hi,cvne a'peets the ubonucleic acid is cyto'ine h some aspects. the.nhonacftc. acid is ad.enii:iu. I: some as peels, the dbom.tcleic acid is * guanine. in. sonle aspects, the method thither c6mpdse qhthes.iziiig a copy of the genhhhc ON A-u IX eD Lit. ..z' t Oil O'' itt hS B4')I ICC a nIsvmc kic u 1 a c' ni the oligoriucithtid.e ia with. a &.ox ribonu:1ek acid seq4eide.
Q4fl72 In som,. aspet Is ftc' due&t"ig 4ep tompn. ci {echng the cops t the i. nom'c DNA-oi.igonucleotidetagconiugate a complement of the.ce.ny of the.genomic DNA- oligonucl.eót.ide tag cohjugath a rdv.eñie complement of the copy of the geriom.ic DNA-OhO;cmnta.Iettkh. tap 000ju.ate, or a. pcmrton. thereat.
{ft0373J In sonis. spcct'. the cop3 ofrht. genotmt DNA uhgoriic. cottde tar onugate Is synthesizecily a rev e.rS. transcriptase. nzyme.
[003741 In some aspects the detection step comprises hvhridizat.ioo of the enomic DNA oiigotmc.leotide tag. cxtajuctate a. coth.pienieut of the.genomic DNA-oI.tgonuckotide tag CCn}thC'C a jet ci e eonlplemcnt at flit. genouiie I) N &-ohguratc eo lee tag omuf ate, or a pcthlen thateaf to *.hi support. 1.0 SOni.tt aspects, the.detectj.n;t.e.* c.o.ttipritas hybr:id:i;tatitin.
of the genei ic DN\. a. eonipkmtnt of:th. >it DNA, a.mvet$z compknei t of the &enon ic D\ \ ar noruon thereat to a sohe sarnn Iii svne aspe dI detrction sLn ttflflf C5 I)brk)L$t on Ot fbi. t'b1fgOTmeI.OndC tag, a compktnent ci the o ooucleoIide ag, a rei:ise coCiplemuli afihe ohgonucieotide ta,, or a poltion meted to a so_Id suppott hi 0? hã6utfthpeftbcgeaontD$# àgonutstMs thi' fernoaotme opoftht SA àfiguást14Og pthUhtsona:th ddtS*ièzt i::: :M: $1::h*n.: Stthc cO ft àtnkDNA3àtsfli of the topy oft genotnia DNA,, a zevenc compkmctuv of thc copy oft genomic DNA, ova &dn*tdp'tybrf6tzat :, có ØHjiflà ojcftWnt*ftlt tbft:thttUckOt1dt 4ttpkW:t1StthlM *iPtfl!1 (01751 tisorn,, aspa$* Thw dcdqn tt cQ:: WW3flggf the genopnc DNAk agisiaetag cU*jugatcc.t cd plo Utàf the gøthcDi&.oljnStotidbtag., p:crnti.atqueSng d* **n3t DNA, a complement of the sonomIq DM a nvern complement eft pturnxic DNA. or a :p4iOfttbettGC ht: taspecdwdetoctton step cmpincquncütgame; sägoaMtatwrnpL*tnt etthe QBgomsIstide tagae:tment:Mt seqtaIrg of the copy otthe genolnte bNAouIguateotide tag coujuga a cornpknn of t8: tcp:comjis uenciag:oflh a cør*P#dk y$fL, 11. IPN&ir*$W*ié äTtbè :::y ôlthc scqocneie of the copycftheoiigonu*oud.s ta&s cmpkmcm of th copyofThoohgonuttcutdetag, a oo3%j in some asp the g bMo1onuc1cot& agconMatb SanpIit3eL lb !orn# t'1 Iixt4ci hSn an ti sad crnpo4tist*aouián4abli4a flkUltte4*;Mt * basDNAimd prçitths 4p4 enzynte4 n scme mo$taaçe 4$ kite aM øornpoMt ions are wed %i' flkSaS moleSt:tan bn PotYe1wW$ RN4 4iS fl. ü *L; tfrknd s*srn1itsc4 tok s4odSaUyI1abetügaDM nicctle., Oft378j* In st,i.nc hi.thricsthe kü coirgwisc a stochastic].abel p.rirnei Lni:rsa P(R.
Øri.met, d.. hilxkd prhrie.r, tevers tnmscriptac. 1)1)0 eizyrne, pc)Im1e][tae, buffèP,dN.fP, IUdc, 1UL srtc ilit P rLI piniie nro o]t,o, r ry cn bma tar thucof \ILt1dL\L the kLts..Onfl1⁄4. 4 4 W1iUA\ PCR pnrnc. b a Cv Lthclcd univetsal PC.R. rr the a Cy3 ThcTa Cirid. awl di) an arra/ The aria can be a 2x8 dmiy.
1'h.c kits disclosed hereih can thither compti.st a stochastic lahd riThcr, cafltet cOiitt{3i ohk:o.
not se tr tnc. rty'i ise UDG envme pok met ase gene' spC\ mii nrtmnet S hi eet specific Irrnuiers., dNTP. or.anv tar nhn at ion theteat [003791 ?) toJiasuc labet pinner urn t.OThp9Sc pnnle'r irbtthLa to <w ot goniuco' "e tag, wheiom th. oligonuckabde ta crirnprises ohgo dl se.quenc aq.nique idendfkr region, fliRt U. nvaTS4i primer binding site, and wherein the universai primer bhiding site can.
en.abk annealing of the un.hersat ?CR punter of the kit to the stochastic.labdprixticr. In sore iSapees. a.stoehastc labe! ohgo di friMej is an QhAi.eeotide tag a ached to Sir ohnc if router.
{00180j w the labeled pinner can uMnfn1⁄4c rrn Libeled v ith the he pirci can be a universal kR primerS Alternatively, the p01)1cc isa target spccittc primer The dye can be a thmrcscentdvc. in some i stances. the dIsc is a. Cvdn h) Snhlic tstances, the GyM dye is a CyS d' I (U81 I li' has and cOtnpo'sflIons tIic losed h. em in <mu-mh ornptms' a pH proh. In otnc: intns. the iluirahty ofptabs i hvltid.iyed t6 the arrv. Tue p tr1hy of probes can ahow ki bnch:nn.ion.01 the IubeHHnolecu}atQ the.arra. The plural jtv Of ol"s can comprise a. sequence that is eompkrnenta?y to the stochastic label align di.
Alternatively, or iidditkmally:. thd plui'aiitv ufprobescotnçriss a seque.iwe that ii.
complementary to the inedecide; {003fl1 The kit'> and cOmnposftiOTl\ th\elo'ed hein ian inther iomrlst one or mo reagents to remove nofi-labeled i oleenles. excess primers, or exeessoligonucteotide tags (or stochastic label primers) from the sampk: conipri:si.ng tat eleci-moleccies.
[00383! Tn. imistacccs the k:its and compositions comptise a rernrse transcriptase enzymcc [he rvTse triscriptace can be MMLV.reverseftranseru)tase.
j00384J The kits and. compositions can comprise, a p&ymerasc eneymne. Thcpciymerase can be a: 1a4 poftmerae. iore.xanipk.. th.e.Tøq r lyctasc is t Ttatium Faq QiYPiCV?$C [0O385 In some instances, the kits. and conlposition:s comprise an enzyme. The. enzyme car i-c an RNase cnn inc Ahcrnatn-elv Ic enzyaic is bDG In o*er n ancc%. the en,vtnc is a restriction enzyme. The enzyme can be a protease, In. some iu:skance:s, the enzyme is. a I*Snzm. AUanathStctheenzymàa gflckbaSeonSSan $iit& :jj 1 t*tettt2itaWab S oydtSSheft{n.
toOa$i j*ø aMJ:a$Mi M: &$:fr**W T$k*t Mtbtt: Mctttt effinency::oftm nftg,ampIxficaton tt* tranitptiot4 aSmo1iuk:canbo axitNA rn&St tIiWA mt1ectilcc*nbcapty*nyiaicd1Mor u*fló* 3.k44t (OOSSfl flSSa flit ton thasoilthupjtt& sóIid:spS can bea ka& the bead can hyki&e to the Iabeled-njolectde Cf ft bead can tnahk detection of tKe labeLed moMukThbeadanbnptaki41n!**xd a hdn-l*hcks Thekivt ithco,thWad 4rthtnt ttti±:Sfor igt swa *::*ts*n tSio q ii1' ki*Wss1t, h t;t khsfutthcr comprise a Thermal cycla. Tire kits can 1xxther comprise nc or nre cDmponcnt ir The oneerna Sncqueunprisc neqtunce% anti nsiiø pths fAcncisteuóinotti:cwthinatkith 4lzaotifying the labeledwtsbsoule The ens' or more conçonenu for dcteeting andict 4Ut*9th:M$4$ó'S SOthP isP asay*tes! ayrSçES 4*sh; s; m$aoI*tth mM 11 44 IO*fl1 $$ifrt4$$$Q:th:$.wä A tOft3MII Part 1, Etrpfibmafliochastie Laithig 1903911 In thia *itep, the stochas* ktWL4 arc antaled to the pety A RNA 1* incteaac tltt à*4 toil oft hi &b aLl SftS ijtfr$& *: t* fifrij 1aIM W::* :!j: bqpzopprt asC4whfl ptpanug extremely bw cenctrnuauons cCRNA. These spec tat tip*t can be us& fbr ptpasfng thekMA;:suIeSatrnsihgns:na I:ft:&hatototth'*iMii need j*i:4:4i*t O$øS $: h*fl:4fl 1 iøS*.1: baEW áis,rtgIar Sbn: 1$Sl N: Wa 7kiL K562 Total RNA. (.ig/ut} Jut 10mM dN?.iP ltd (enc SnekltlC lt P Piitnei (1 Oi\t) 04 Stoch ts Labek l0u> )4 U 4 ii iot& 106nJ EOO94I Md 2..gi &the RNA mpte tc be natyzed t!}Q35I Mix welt)y pipcttlng and spli. briefly [OO$t thu. b21e c1 ft toi meuucs Projam,ind then riace the tubs on ro. <ft ieat I inmate, {t!03971 hi this1 atep. double shunded eDNA.is created ku the. specific gene. of ikiterest.
Each cON A r.n.&ecuie Will now contain a primer site ftnthe subsequent PCR. step. Comhn.e the foilQw.wg to make a matef mix fti rewre t.ranscriptioO: 5.X D:irst Strand.BjrFIti 4a! flit I at SupcirRNaseln (200./id) .1 tt NPvtLV R liii NEB iaq Polymerase OAj.il.
The:u.n;tJMlf' R 7? anI VS IRaq Po/ymeiwse IuA.iead iSqiersc4pi ill and 77tanhim /:/ bcusea 1003981 Add 7:.4n4 Q r aster mix i. each tube jind. mn lw ppethrm g3tit.Iv. Spin onelly.
1OO399 Run the {llow.iig program (Program 2) on: the thermal cyc;ler: 37' to 60 nuectes 3 cyides of: 94' lbr 2 minutes 55> ibr2 minutes 6I: for 2. minutes Then 4,rever 1004001 &IL.i the P( R tWon. t 1\ ttCLC5'4 to cgct the sanpL with Utacu D\ \ (Thrcø ssc U DG In pn \ en{ c naLorponed r Cr 110W hcnn anphfied in the eene spetdfic PClt.
[OO4GJ To each reaelio:n add 0.5.U of 000. Mix very well by pipcUing. Transthr all liq nd to a rew PCR tube to enw c tha there i no c. ur o\ er of unmixed 5ctflLL {00$02J incubate at 37 fin 30 min.utes;.thea 4' ti: t»=. igwainacR 14$t, : a makamaaterS%rPOt *941.
at Ta Buitt Ii 3fll !M 4NTP $:S:P 0 fl.M)
NEB ISgl
{51 Fiursi cøzwentr&dion OfGOSnM pctmc incrcasn spectttity otpm4ucts tN4uq Md5a ófi 1:. SfremthprevIst4toanqw CRh&M4 1$pJ flUX to bth Safl*e [%4fl'7f: $j vSfl by E4E*ttiót 414 $h LWt*.
n&tBOwgprDa(Prsnt4io*ttrrneJzt *4G fálmtos I3Occtewot ** fr2ganows sr 6*? 2 rnhiwss ñettii'fr4 niMuts [4Ø, fl::3. saSdk [Slot iPthctü*WThtt1tS1$1ticsh4PcR Nuckas&tc water FOX NfiBtafluSr 1 5it$UØ$l:PcØtQtflfr xd :NEB Taq:Pmeras, :O,d Tt1 4.
1WI* M*W$*P*$f!fl$$$$ tbëó *E44 vS. wSnpistg tnid vS inatioaotthe pre4'CIt area.Nrsn:aU sbquerit Meps in this area.
{0041 H Mi seU h nmertrn and pm biiefl\ 11004t41 Rth 1h.thIIk'wTifig IiiLkti 4) tt the ihdrma1cyci&: 94 *r 2 minutes 3Ok'cies of 94t f3r 2 minutes flit 2. mm ütcs &8 ih.r.2 rain u.tes.
Thcn fbr 4 mintnes 4tbrevek {t!04151 Option& ;Step. Run 4u.of PCB. prqdvct on a. polyacrytamide 42c.% gradJent ThE.
g& to assess size and purit' [0.04t4[ ?ar 4. Target Hybdiatoi.
t004171 Taru hyb.dven On at.
1004181 Ptcpan. tpe fin hinchzaUon to m Anpacd \1 eLOth\ the. rnty nde Ade the ollowhi iRa ft2mL.PCR tube: .SJtq1.A.(1X. SSPE+0.Oi Triton IX lOOt 5iu] C2 Chutrol Oho (76GpM) ld PCR prod act 20u1 Totsl 76ii [00419 Mix by pi.pettirAg and spin. briefly.
[004201 Thdubadtubes th 9& to deiwtua aid then $ac:e on ice 100421! .R.emeve adhaaive seal trotn AMI amivshd.e. }>jp each hybridization cocktail flt) a eu of the k\11 array %hde \tiL * note ofthe erd.r n' s bich the ngs are audec Cover sthk with second strip of adhesive. (included) [004221 Phtce sealed array slide into. kumictity ehamter and çj mb hybridization Inc bdeat ocernight 00423.J Part 5. Array Wash. aid Sean j00424J %jjr the O'veflugtIt h)brduutu)u lcd e he nTd) dide nut o dir hvbch, £ton ocr a ICIPOW aihene CO\e Ppetout cmamng hshndL7aLon coextav anc save at 3O if desired.
{004251 ICIispeisc I 50p1. Wash. A. to each used..:.e]L.AiraOt liquid and disdnc I SOp! \\a' t B U CX sSPEfO 01% pion \ MO) to eaci efl &pn.ate hqwd and bnn ara d Eo C*Uiflt i' S ihC arrays wd ibese atoned dry.
1004261 rn oil th Seiiscvatiri. FLAIR iPStruient. \Vt i)n iu.te fi,r: e riacItin*e to wzikn t.p.
iOOti Open thetofiwarc..a * nd dickiray.C.Ipetf'.. Place the.mysIide into tbc 4-1ik Folder. Be ure tr scaitbe slide. irôei1y. lb. the sOftwa.re.i.iick. Thty C]óseTh ioft42J Click the Scan!' icoit. A window.anpears.with infbrm.adon aloui the scan to be \1ocl;t}he nThw ofthe san 4 dc"utcd md ec the apropncilc seth to be & a mcd l, U ck 11W ii t 011 Ill 1.1K S f51'i lofI'c lie hi Ick cciii v ed thai 1k to K. scanned. Tbe soffwtire will circle each stilecwd. wtli in tdllow Click 1904291 e Plate O.ec. c' ". it dos ifi apptal \ho\ mg e ptoy eS', of the can 1. hice a well is scarmeck.the. coior.on the screen wilt turn from grcy to groat if the.rcfbrence pa.ttcrn has been detected and thegrid has been positioned: If the refitreuc.e pattern has nut bear ddtccje4. the well will ho colored red. ff any ofthe scans do not ddtcct the rcftrhnce.. We grid cww be ri uel ahgned by c1ul tug the ieanai, c buflon 3t Jli, tOt ot the u-ei Thn i P d:ispia.y the gr:kf which can be ios.itiOncd p.topedy. Click the green Thecept ysis button, at the top of the screen..
IOO4LVfl Once all ofihe grids have been.. aligned.. the data. can. be.ext:or.ed.. To obtain windows fimbtiOnfflit' ptecthe indbws" key oh the kbybohrd end "IY' simultaneously.
ILo.cate the Scan rc.snits in the fp)S doewnet' .s" drIer under \ravkecidc.. noauon anaMoader eameiults Open the' app..upnate fokiet rid copy the TtFP u.na es inn! the result.csv fThas. to flash drIve or transfe irougb.the network.
10043.11 Proceed to data analysis either manually or pith a computer software. çackagc.
1I0042 fixp/è 2. Four apeuirnenti if here /20 RN4,wieeuies weni added to a qpj' of bac Lgwntnd total B \ 4 {00433J 240 cepte' o. a potyidenlated nueknc acid hagn'enr ia ciJdcd to ci 10 UI reaction containing IX tthmium faq DNA polyrncrase butTh.r, 0.2 ii.MI dNTPs 0.2 hM of a pool of %0 oligo (cli) stochastic khcls 0.2 pMof a second strand eI)NA primer and t.2 tL.
of Faq DNA poi9nnerase. in some reactions, an adthtion.ai p.um.hçr9fpolyil.derlyl.ated DNA fragments with Scqi.iCtlCeS uni'c:latcd to the 240 copic. oft.est nudcic..add:fraroi era were.also ad.de:d.. In react ioo..A i.xi:0° baekgrouM. pohadeuiviared DNA molecules were. added.tct the retiet.ioh. In react4':p 8, 1 d 0 ba:ckgrou.rd p yitdcilated DNA moeeutes tyci. .ddcd to the reaction, n reaction ( 1.5:1 0r background poiyadenythted DNA: 11 oiecu.les were added to the uthction. And,. in rchbtion fl, no haclkgrouhd polyadcn4atS DNA. molecules ecrh. added. to the teaccon 0 mci I rig or ftg cif iauaornL frisjnented an pohac enylated tiI4niall 4,,croruc Di4*A was tested. Afier 3 cycks ofincubation t 94 0ç< .tor 2 n.dn. 45 tt. ibr 2 mm and 65 °C for 5. ts,:1 unit of Uraci..! DNA4ycosviase s ad.th.d and the reaerihm Is incubated fM' 30 thin a 3.7 "c. Half of the.eiitPon 1 thea ad.d jiç a.30 i. L PC r6aqiofl tti: \ 1 itamun iuik. 02 tM dN ft 0 jiM g e-'peeifc forst.a'-J mmer. 02 uM a rtiwrsc pririicr amd 0:3 4. Titaniuni Taq po!ytherasc; PCR. cthiditio:M' wre 94 t tbr 2.'iI 4R&wc:dThy 30 cycles of94 O(i fr 20 sec. $S°C the 20 sec and 6$'C i& 20 ace:. A final ircbation at f. °Ctbr 4 Th Was perthrmed. A. uested PCR: is tierfbrmed tbUowi.ni the 521r.fl.C cotubt)ftns as the first.PCR, except that, rested kirward irimer was jise.d. 2 iL nt 1:25 ditutmoji of the. initial PClt used as te.thplate thr rho nested PCR. PCR products were a donik trakn uteC it DNaS b1Otlfl4O)Jed with I entu Icil t'& s,ktat.e uiZ'nn and Own Fi5hridized to a detector array for 12 hQurs. atd.Tt. Signals from hybridized were deteucd ta taw.n° th eptavidni eonjogited Phoervtbeun and lflkU&It} on a miciqarra.v can:ner.. FlU, $A$) shows the signals froni 4'ridIzed I)NAs tot naetion.a AD.
respectively.. The m tuber ef hujids p.rese at in the. trebrj:dlzett DNA is count d and used to ctetenn ae he nuinhet ulontural copw' ofnuelee acid tian eat' Reaction it of:abctls 4 nt ormulal cOucs A P2 130 c: 109 11.4 Ii J434I Ewrnple 3. Cnqiartwn a th t:tai PU? jOO43S Tie cnn ecatothon of u a' tro athenbed R'<A w-a' deter nired i'>irg n \uder4 oaaaI.zei mcvutnert 0 jiz o the R. A sc mixed tt nih 2 ug of a K'02 cell line t0} RN A t. ha It v a', used a a an icr The 11". A it raur in t I V\% .1.1W.1 a iii of a 10 mM cr's ft soLtho a and 2 aL ofa 11) a Vi pool of 60 ohec d 1) lat'e,.' anu uL nt's'. ate4 Ths nixtuYe aa incubated (,, fM' ri d l&jii1të .çhjflcd o4cë 4 4. ofafirst strcnd teaetion buffet <25OmMTri4-HC1 (pH 8.3. at 2&°C). 375mM KCF}. I iL ofO,] M DTT, 1 4. of RNase inhibitor (2t a it) and 1 i.tL of supemtnpt 11 reverse. transcriptase (200 a at',) v as added ant. L1. eacUni. c a. £itt barcd at 50 C Its 6Onin a id then at 70 t loi 15mm. 1 RL 0.1 kNase ft ii units) added and the reaction was incubated at ft in 2Omi.n. Digital PCR. scas used to 4ua:ntitatethe ntimbdr of uc4sie Of eDNA st.he.siited*bro the; in vdro traMcdhed RNA. The snur4ile was aFo rest by stochastic tabeFiug ?CR 90 copibs :tthccb$j4s4óicrmMc4 byd tPcts4ddè&kn iD4scSoneoaiainbg IX oSsteteaSnwos thjubatàtti t*144t%rthbi st::mn:& cIa :Lg1tosdwaaidetat14 Srtatiotts inSatd at ttrttomät;fl* and $$4 q::fr. 1* 1. :: w:*tn in PKL3 *ow ttmtttltinbe *yfrj4ThNThna*c taflthttdhe bA':eSitètmiànmtot ót&i ofl. 4Lidfr4$øL44::tYi gji5iai,. in hyb4tN4in4 41 snttaSb' stoth kbciins:acb*ned to a tepics atntttby 4itM PCIt ThAse ritsuM danoztgfrate that sbhastk labo) jn an ttht method tt *Sn*1. tkceuM S Ski nmcy 4$ cpnpa bchflisi Ptct iE4i:. arnpie & iM&o* sftA:k S [*4!1f: To.ts:the ØtU.Mà 714 $MI'. *iñgt$*'$ e::::i Øii sanmna dutsnnpSth tl4Act*X k*s 4ath rarMi eøpit** an n vitro transcnbod pØyadcdylated LMA was tcMcd àtx stoczhas* M'eLm following the :ptoiS d,c t exsmpbt MdSnartly. atkIAjob4edtrn warnmalian.ctils.
M$2 itsg: tWA were addM to The reaction mbthtrc Each r4ackon uS anywben between tS gto 2 pgotoankr RN& Ths tgtRMn*keu1enmt.Ssthbe4 tqP>1A wn 4 obsewtt Attbeta tt$**ke ar;:s1 køa:s ih5 0:JflCsdt4t nctRL. SscrniI4 be 4b:tó ztk Pfl 44i shówsti :gp tab Is $$i 0
-
:C, :y tVQ:, I P F -RjN[* H ______ _____________ 1 ___________ 4 ______ [$4.SI $c$$!=Ø$ flW.4V*WR*IIf$. -ste
(414; The Oefth&\itatMLV M *itti*ó &Näifl MVvS aL tiij flflôt;I$ 1ttm tlinu cd potvaucnskxlcd RhA dtkIZkI to a (4rne1 CL I ji tISIJ K5 u. tOtJi RN \ h: RNA', w:r added mto a U 6 uL ietC ontort9nm I ut ol i RhaM ciN ft c[u1ioi 0.4 1tLota tO tth4 ohd sthund primer. ti:4 n.h ofa 0i4jciol 61960 Oho:(dT) eis, The reaction was mcuhated ci 65 t thy.5 miii to detrtuie the. ItNA:nd then quickk chilled on icol 4 aL of a sx firt sltadd haffrr I 4 of d OhM fliT, 1 4 éfsupdrasc RNasc* nJLb1t&fl 2 umt.') and 0 4 iI at 44 DNA pc.$ cmc ase 2 una as addcc Additionally in A, I nI (200 umi J of t} e RNse H rru mift m po c p fli) is added And 0 L.aLflnfl B, I iii. f200 in its) u he wdd N \f\1I \ L. \st. LI an',. i',1ac a'as add d Ike Si CIC hflU,.hcild ii 12 C tar Ornin fdlloec b 3 cy ie of 9 C' o' 2mm 55 t' lo 2 turn and ñh I fbi 2nu I ant of nac I) 4 \ RIvcn', ase was added and 1 iLacUon was mixed and moved to a new tube and incubated at. 37 t for 30mm. 5 pL of the reaciion was then added to aO uLPCR.reaction consisting of IX Thanbim buffer. 02 jaM dNT. 0.2 aM gene-speeWe. I rward prithr 0.2 nM tmhversai reverse prun& aSd.O al Titauiium Tap fflras Pfl conditions 94 0(1 mm fol nwd In 0 ks,. L cr94 C nr 20 \C,, 58 C thr 20 scc.and 68t tOe 20 sec. A final inctrbatioitaa 6:8'C.iOr 4 i.tiin: was perthrmed. A nested. PC.R is performed following the same eond.ition as the. first PCR, except that a nested.
Fo ward primer was used. 2uJ...: ada 1:25 dihitionof the inidal.PCR was used as template thr the:nbsted. CR. PC.R pttxlucts were randonily fiaLmerite:d with: :DNas$ hiotmla.be.icd with Tenr.iiima.l n'ansthrase ezvmc and then. hybridi2ccl to a detector arra' fi t2 hours a $1 inals tram h bridized DNAs were detted via ste.inifl with S'treptaVidin etnjuttted Pbyeoerytberin and unagwg on a niid,roarrav scanner. FiCi.. 5AR sIu.wtiw iab&s pros eni in the. h brichzcd DNA in reaetionsA and B, respectively. The nuinher of labeth present in the hybridized DNA: ii coubted and. ubed. o dbtdrmine the,nueii.her of origintil copies of iwcieic acid fram.ents.
Reaction t Input INA Reverse Tratseriptilse #OfhtheIs molecules A 188 Supetsehpt Hi B 188 MMLV: 124 [004401 4mpk 6 &impanson o/ potrineraccc to er rnd stnrnd s; 100441! K pedOflP4flLc oflac i:ol.un ie sa. e.mpaied to Titanium hip p4'mcuse 1 875 copies of an in Vitro in.rnseribcd pt$yade.nylated RNA was added to renedon A. I 88 copies of an iii vitro tra:nscnbed polyadcnykitctl RNAwas added to reaction ft 1875 topics froñia K343 ocR Ike wa added 24 de**ofthere*ctk,fl *nktua Ths RNAswtrflddcdu)te a 12 nto ii uofaiomMdNThduttt &4:tLOtt 10 jAM iiceonñ * SI:cc4 gLti 10 St tei96Oli(flytl Th*,***$4 at 6sc:i:r &nJnto4èm Th:RWA:az4thaqukk1y SiDndoab 4Lof Xflr& s pLots ohff 1.4:thi4as4 flae inhthtor(b U1tteste i:*Sii: I Ta zithØ 4ó*aiä ml kS4tC r 2t for 2 nib and 6*t far tka I unIt of uracft DNA glycosyla* was 444cd tod the tc!=tMu WW:TIb4fldi1Q!edk,, ft nw tbean4mqucq4437*C tar 3OSi $L, td B %vdthiAed *ith?a 20 itPC iraotjoza.o gofljbuIt*1Oi uM jifi:: tptht OUM 1p*k$4j4flq: DC Tkaruum bi4frr 04 M dNTP, 42 tiM genc-specxftc rwardj,rtma4 0 201 umnnal imcpMjLT4amflq;kcox4itsLcwac4tt 5w2t uwD*ttrasOc Sttt2b saat6Stt:2On it :Ø*IPi4 $t3 fr4l. **Ø Ah'7:cRti4ia thiüjU*i* :sth.4ast&st** ex tanSdPovwacdprincrwanseti2 jLthii4: dâhuiott of the iáiaI PCR wa used as teniplate Sr the nested KR PCR. prdncts were fflMGJ*ØSWWM* WaS-Mb*d w STS4àJ4i. :4i:y hbdaisd dtcctotaAt*yt inni1t: SnS fit hflr4&t&DMntt 41Ø*,, i:iad:E::saaiths, s3:s a :thkmarmysariFIG St showstibek presenLihbD1M:thrtaethns A-A rtapeawly Thc number of hbth preseM in the hybridized DNA is couzde4 and used RMthon 4 I$put:JkNA: #OiieI& A:1*1% itt -S laq 151 :1815 Thai - 1) TiiSrnTa4 421 OU442j t\ampk AMoIuw quanwatwn o/ rnR\J b3 toantutg vthwdued D\ I rnoieckc UO43I.oiR. A eak be by fh& ackIton ot hds pr.t aniphficiiica of E)NA mokeuks. ( 1 -JC. I 9). i:abc.kd L > DN:A n keule.s are thrpied icy.0[yii\ synths of an mRtA nm1óu k by the ad.ditio. ofa.*t ide ptii.er v:tth (i) an oj 4J L&P1tflL( tO annca tO tI, roy \ RNA t iii (2) a LOhlCCtiUTi ntpicdeternincd fl afltIt3m sequen&c ahet tag'. and fl a urwnor o urns er?1 PCR pnmri uuee The boded ON A nicvc&uk <uc a up U'Lcl odin' cncspo. the p irk N. id common or inm% ,s'il PCR iifl%i After arnptificatk%n, the numbet of labels of diffcretu sqitenee eompsition can be readily t1eeued b h'brithzanon, eqt enrn' or othet Jetceton metoods 1 he ddTicuh a4 ot counting the number of individual.mRNA molecules in solution i trunsfbrmcd into thç simple task of determining the number of types of diffi'rent iebeLs each being present at high conce flatiun' toJowig mpI I icanon provided that ne uflJ label equeree dn Ctsu\ is sutTh s, nil l giey Ion the nunh i of molt. th. N.mn' orhn U Uf4lC nb'thOd i be used tt incorporate ubeis into the KN.A orcUNA molecules befere.or dtuziug amplification. Any other.PCR or noioPCk based methods canais:o be used to ampUfi the RNA Or eDNA. mn.keutles A]Thcunh hciitul in thtso, exatnples. am ttarstaati nithe. labeled mo lee iles mdv be ibqu.ired fad detection.
[00444.j.Erwnpk & .DigiSinicnuiat4vfrr.RA4 oprntian P1w mRNA s,e\ cisc traree"tbed asilig a pool of n okud I hthe prime p andom ilo label' may th,o h us. ii FIG tth Inc DNA eai N op{mo niH ami ti J ith methods such as PCR and 17.ampllfication. The labels arc amplified along with each dDNA.
tholedule, cONAs. arc. hybridized. to djital attain to deithnine the rwntbedof distinct iabeh' fhc each. gene. of interest.. 14yb.ridizat.ion:recjuires both p esenee of iii ore sequence. most hkel3. scg'nen on th., 3 econ at thc gene and on: at the labc sc4p Ci LCS An arr< , uth million features is sufficient to detect acoHecthn of 350 1 bets apphed to a sample with 2.OMOO different mRNA sequences to determine the number of copies of each tiRNA present in the sample. A subse:t c<f the 350 label primers may be appiied at a lower c.oncertration to nereasc. he effective. dynamic nmge of measurement This methodl ispamt.icrd.adv hu saruphug UflUlmflg dkOOUTItS o OathiHZ 1PcC'11cL {* Iii snufl t,elb 1004461 Euvnpk V D/'rra! mwwan br D\ I tvqn' nuin her [004471 Genomie DNA digested nth small fragments inane, or more reactions usine one ar irarre tfjetjod eazynics, Adaptor,v.ith ldbel: seqttencea: i3e liated to th.d DNA thinthts 11Cr 2 I) the hatcd lraenwnts ate opuonallvamphfiea Ligated ftagaients nuv o be digested with one or more rcstdetioti cnzvn es prior to amplification, to preen'i the replication of certain thurnnts, which. :j: thetIti in th iectwetnp.iic.atit.ofonly fra ne Us a intces{ f4 n d alion u chgtcd atra\ S ciuect ik' rut the oIC r4ifle{ lalit ts iated to earth rc.st±ted fragment; I sing'iO}abd'seq.uenc:cs. an array o17 million &ature.s cart asssy 20000. .fragiitn In the geno.me4. which represints average inter'a1s ct 1 50kb in ieai s some allele speed'ie tIaenicnh ma be a,'aed by dm..'s.ne tneton enzymes (eg. 4 base. cu:t.ter specific Le an allele of thwrest, RJO$481 I vt:rnp& 10 Digital niwi pcura for [004491 Labis ate attached'to the Y ard 5.' ends of microiNA by ligaticin or other mean's IC 22j I tK Lthd.-mic oRN \ con pie'. s et'1c an"c ihed to gene tie tbel4YN \ products... The la.bcl4.NAp.rothwts are optionally amplified. The ibei:4ThiA prodw.s're hvLndh cC en dwfai array to det:u t mnnt e of labek per nncok\A nnRfla,c 18 (htp.://ww*mithaseag/) was. rSased i 4c»=vember 201) and lists 1921 unique mamre he mar. rn.jRNAs. An array:of 2. million features can adequately detect bOO labels. ligated: to the. Th.21 miRNAs Dft40J Jr srnpfr / 2 Dg:wJ nnnroarn for.sng1e ee/lpie-rmplantatton gsnnw diaEno5P [004511 Phmary h ehfr atsingic.-oefl gehotnid PINk aiopiitcittioir assays is thnt'allcle C il anc i Ets'a huts \s UI tlit COP pdtctt KIfl inotit?tii ck si II C Where every btok.culC juts a (L.S probtnihty of rc.plièarior. mo' uies tf 1:1 huitioi'.cpy ratios can easily be distorted to 1:10 or. zrenier just alter a frw rcrpRcattpo cycles.
l0042l Flowevu. %hcn kht.k a.t.e first artaened priol to.aniplIheat.on. counting hthci to detcnriihe copy jüir±iber'is tm'aff'bekd by Eeplicatiha bias, so loh tk replication oceuric \t ouh. iii, does not sok e I L 1uoblcrn 01 alieb. rhopnut\. reupknds di tenmuanon a at eec gttis adele on ot ampnaatior1 n be e''J) md stctnate1. detununed I bIN 35 particularly useful for PGI) applications.
1.004531 Kromple 12. *Diqital mfrroa.rnryjbr eneciawhtgfrwtl ezn'euploidv in. MofrflMi circulating nuc/eic ac/ds Dft454J Ojixitall nhiemarra can he' nunir fetal: aneupi.oidy in m.aten;ia *l. circulating Itutuew': at:id&Asarn.pk c.sm,g maternal cwc'nlttttr.g nucleic acids is provctetL The DNA is fr&gmen.ted U5iflH5 4 base.eutter. Labels aP øttckd te the fragmented DNA. (:jyculat.
and muhiplc PCR to amplit\' 40 chrormsorne: 21 markers and 1.0 c:ontml eromosome thhr'kcrs, IDteet athjilifled l.äbel4)NA produetson art array of'S mill ion featureS. The nwnbôr ofcopks ofchrtmosome 2. can. be used. to dcrm.iue fg occurrenc' of:fet1 ancupkMv (FIC;.24).
M$551 itvwnpk I3. 4cohj/, qIi(IULU(ffi.QJ1 t/mRi\4 kr c4fl4nhii!g* mndWuqI i.N4 moMcuk t004561 mRNA. rnO.k'c.uks can be quaittdd b*i the in:cotporátio of 1ab' .d.uüiI, tint strand ciM \ sy.ath.eis (FRi. 25). ]abekd tDNA no keu1e.arc iormcd.hy eDNA symhesis @1 m tr R\A nlok\ %do by th' dduio @1 a dco yokgonut lctmdu pnmc wftb (11 efl ohn tIT to annea k k. potA RN \ taiL 2 oflcctor Of pLdt kimiuLd 01 ia; Join equenee label rag and (3) i common o non etsal PC ft pumer sequLnu Attt. lii, snatd el)\ \ grn the numbet of I the, ot'thffutnt equencc co rpoi ion Lta he i aJ ill ctetectcd by hybridizaticin sequencing or other detcetion methods. The difficult task of counting the number of individual thR.NA. molecuLes in sohition is trans.tbrmed into the simpló tS of dctermththg the.uibe of tpes of dift1ren:t.labis, eaa. bg peiu at u m nt. at inns to Ion ig impl tk atu ii, p 0% tO> I h it h ifl ii label serit I CC do i sit's is su ificic tidy: greater than The. number of molecules present. Any other suitable uttbo.d can also be used to incorporate labels, into the RNA or eDNA molecules before or during firststrand..
cD.NA.iwnthe,cis.
1IGQ47.i Example 1$: Ii.tkni pnë with: scthl dilutions ci kanainychi RNA.
RH14581 A titntuon..ctnve was generated. liv pcrthnr:Ing serial dilufloas ofkanurnycui RNA: to illustrate the bd dynnic raniwot the:abso lute cduntir.gprOtOtci:. Each of 9.stri4l dilutions wa normahzed to a conceniration 0f0n fi/d tl'om2,5 pn&gl. 1.25 pg4il. 025 pg4d. ft1.25p1uL (k025 pg4tt, 1.2.5 fr/j 2i ig/d, 125 i/rd.atid: 0;2:.4d. All ofthc Wiutions wert/ruade tsing..a dilution ohllion at lm&1trl E. C'h totid RNA in tubes drèrdiscd.
wIth..a so uin tot' I Dug/ni east..RNA.te hinderthe.stieking t'f hesntpfr RNA tcrthe walls oltuc tu\ The s'ipin >j( iddei a:' 12 (d.ac on cont>uuln! 1 U4 F Colt.otA RNA I 1j ola 10 mM solution otetNiP's. o4J ofa IC u/VT U pruner specific forkanani.ycTh and 0:4 3d eta 10 tM poi of 960 di obgo &hek The reaction was neuhated at65 t fbi' 5 ado to cenaw'se the RNA, and 9; qnUdi thuled an ice $ gE eta SX tiist strand bu c's I ti1 of a. ft IM Dii. I gL.of:superase kNase inhibitor (20 units), 1}i.IL(200. units) of the w:iid.type V t; i trans. npt se a"sd 0 4 ul o F' q DNA p&\nerase 12 uruR) n adds. d The reWiotis svepe inc.ul*t.5d at 37t ibr 60.rnin. fOllowe4 by 13 cycLes of 94°C tii 2riijn. 55°C.
fbr.2 miii and 72°C liar 2 miii. I unit. of uraeil[)N A ghrcosyluse was added and the reaction o & mixed an I rnesei to a ries tubs.. md neuhatee at V C to 1Omir' 5 a I ot ide ieictio was then addedto a.20 tiE PG. reaetion.eonsisting of' IKThq Reaction. buffet 0 tiM *io.l dNTP 0 05 nN c c-speufk Iflt%S ard ponu 005 nik1 UU\ rc ptunc and 0 3 1 I aq polrnua,e PC condsons c ci e 44 C to.2 in 0 FO 11.o" ed ty) :\tle o191 C cir 20 sec. ¶8 cn 2C sec and 72 ( in; 20 SCL A final met buuin a 72 C ho t U1 k\ as pcrlbrrned. A. nested PCR wts pet tb.rmed usin a nested tbrwerd pnmer and: the univenal rdwrse prithcr with it C'3 label atta:hed., 0.5 3d of the initial PCR wat usdd au. tethplate for ihe nested. PC. ?C..cnnWtiois wre ftc satte as ftr the tint Fck cxccpt that. the: 58C slot) was performed a t SYCI. The sainpies were hybridized to a detertor array at 3TC overnight and scanned tat. blow aLa J usmu a fluo; eNccn C rty to detect v Inch po',rtion', on tb.
ae & tUlle mcd t*b C 3 \cibcl the a m17 of po0 a e s. s s u'a d a d 1 erir ne h. n 1 concentration of sn p1ev FIG. S shows the dilutio.a scheme. P1.0. 36k-H *owsthe scatter pletstfrestiits and Tbi. .1 showsthc tesults, FiG 3.7 shows the cortebtion graph.
Gft459J Table. I
[ PIG Irdt.ith c:n.e.etratioii Dilution Fac.t5r hpcc:ind (tiint.ctaai. Gown 23 pW4 10000 130 {99 ( 3 0.25 id/ai. .1000 130 170 36D 0.125 p&SLIL 500 no 2513 730 3SF.123 fghL:10 130 H? 3SCi 2..5fg/gL H 130 95 1 13.0 137 jOO46J E.ump.le:1:5; TDnff ion. eperinwnt with. sntd dilntio.s of hwntn. liver.RNA to measure (APDH epresston M4Slt A titration. cune wasgcrie.rated ty pertorimog serial diltitions. ot human:ther to0i1 RNA to idusuate the abi n of he stochastic Iabehntt protocol t: Uciect L'sch ot enc expression, Each of. .sernd dilutions was,nncmahzc.d.toac.oncntration oil.25 i/tI. from 5000 pg uL 1250 pgul S00p td. 125 p 4d. 50 pgtu 12 5pl S p, plaaa 25 p. I Afl dilutions were. tna.dd usthgn dihiti.on sohuio.n of.! ng/ti 13. Coli total RNA in tuhd p \-il d;th a,ol mon mm 10 ri/p I yeast RN k o htidc hit' sEa 1 mrg ffl tin vp1c RN A to the wall' of the tube rhe santple s.rc added o a 12 $ i I u.aet'on conb tntr. I u P Cob total RNA. p.1 ofa 10mM s<;hitionofclN'fP's (L4 d ofa. 10 nAt dli:pT.r spac.he tbr GAPDH:jjj 0.4 ifi of a:i 0 p.Mpool.of960 di olio labels. The reaction was incubated at (iS 1.02 C. ç (or 3 tnii to.den.aufe 1te RNA.and then. qukkchilkdw ice. 4 iL ofa.5X first tinu iTatlftr, I p.L d:F a 0. I M OTT.. uL of suetae RNae. inhibiwr (2(1 u.nind. (flflQ ini) et: tha i1⁄41 t3 e MM \ ei ue tt ns t and 04 iE or a " rsc &2 untti i'. a added. The. teactions were rncuhatect.at t9C t £0 nun, thilowed by. 3: cycles.o194 t fix 2thiu, 55 tibr 2 itmand 72°C lbr.2 thin. 1 tddt dfuracii DNA.glvc63yiaic*as added and the reaction was mixed and moved t&t a. new tube and incubated at. 37°C thr 30mm. 5 tL at the reaction is U en qcticd to 0 ul PC'R wac ori uriotrng ifl \ Taq c i Non b, O aNT dN I P 0 0 uM cc uL ci h fbi i aid a na 0 O' p Nt an rsai re, N ui i and 0 $ uL lao rohT.cIac PCR conciticnm \\crc t lou 2 nun fo3ew ed tn 0 cveks of9 I to 2u see, 5 < ta 20 we ane 72 "t fs 20 sec \ hna mcthauni at 72 "C tb 4 mm is I' perfornied.. A. nested PC.R was perh5rmed using a nested fi.nyard pdnier and the. universal reverse prime' with. a.Cy3 label attac.hed 0.5ti of the initialPCR. was used as template for th. n 4ed IPCR.. .PCP. conditions were. the sar.nec as ibr the fini.PC except that the 58C scep p a'.q ed c. 5'C Ike ainpk s \ h Ii' nh,ed to a dctect.r ai at s70t o. tn hE and scanned the.%itowing d.av.usihga. fiborescenee r aderto detect whiek positions on the ariay contnned the Cv3 kibel Ike imambet ofpo'nne >nots s awd to detetrmne the urn a ean.eentradcnr ofs:ampk.. ..FIG. $S &:hows the. d.iI.utioim scheme. flU. .3) the scatter pi0t5 of results and Table 2 shows the. results.. FIG.. 40 shows and. correlation, graph.
lIO.Q402[i'abk 2.
39\ 5000 pgd 1000 /3 1250 p. iL 1000 63 500 pg pl 400 63 301) put i 4 a5p.1 2:5;. pg/&ti.. 3.7 iOO4Ei31. Example 16: !1easmen1ent of control baSrial gses [O($64J The t.totocO was validated usTh ?oy A. bacWdai bO.ntml RNM (liys Tht. Dam ithd Phcd as weff as RNA born the Kanamyeiu resistarec gene. 4 d:i.fkren,t. dilutions of c.tc.h control wereused to validate the accuracy of the counts. liar samples were added to a:12.6. p1 i eicnc'n cont&nmng I F ( oh total R\A ul oft tO mM \otunon ofcP\ 1 P, of *I 03 j.:.5J' 44 4tj t1wkNAth ttilSi i4, 44tót*?VUt* : qfl'J ør* i £2Ot3⁄4tfr41iL 2Othiit4iftbc w;.MMtstsea&4L$tq UNA$ittt42 töitowcdby3 qycia:ofl4t:br2ntii, $t it I othittwtaciE b*!Yt*sYMa **Sdeiabd SaatS&wsmt*et and tfi*ewiuband ucij frøO:*Tifl$ cbntuslinof IX Taq aenio buSt, 042 1tM aNT?, 04.05 idA geilcaspecific finwanI prixtit, G 05 pM ugivefMt mvert prbtts S &X pL 7q polyi*msc. PCR. con4itfts were 94t tbr tmIMhn4by:SQ:cyJsofWC fi Zbiec. Mct::tr2O ii1ci&nat72iflfl Anest t*sptuinflnstd t44sa4 th$4$$fl 1*itbjC:e1it*ok4 ifl:M* FCdntrniWttrThss*PCt K dStweittswnths ittkS do ttiyat Xl t d tchpeiUnson*:snay Set. thcnwnktofpøshiv*.ps *St. to ttt St jjj cona>ttstton Saj k:EI it 41, t" sattE" $otstfttS 4ilu$ps :f4Njt$4. t$:bM
citaSen topM mast:tepIn, (nnuufavtp$t) (CR) measured 41*. Ls9subtiW4 t90 41? Pk:pc*!h) In 119 41C Pht(asSlL) i& ]i6 41 t 7,,: 0 #a4s 1434 42 kAtt*bx' 756 520 gent: OO466j..Exnnpie 1.7: Cornpatison of qum1ifiStion of lunamydn RNA. by Mochistic.
bbelh)g flU dgliaI flR JOO46I I oui a rka n' c R\ \ e 1eiatcc by,nciaThc thc' nq e. cmx eL tO tht cOuut', obtamca from J'tL I PCR i anothcr _4'ip1e o ahd4 lo tt R\ s is Ljud to, . p1 rc(t)n oita nni 2 tg of F ( o totM Rt' A. I ot a 0 \1 solutg>n d.fclffl'P's* and. 2 r1 ofa.10 uMI snh hon. of 960 cIt ohgo thbek The sample was heated to °& tot tnin..nN, then chlkd on ice 4 at of a 5X f1tt nand)uffe, i at of a 0JNi. DTT I ph ofsup.erase. RNase inhibitor (20 n.nitshi p..L (200 units) of Superscript HI reverse tt svrptaSe was ad led to the iiadiUn., The sthiple. as cubated at SOtC for 60 nnntttes then heated to 70.C* ft:r 15 minutes. then cooled to 4°C.. 2 units of RNase [-1 were added and the sarnpk. was.ineubahxi at37t:fru' 20 tnimites. 2.9 plattE. was added alter thc tina nuh thou \ 0 nulbon to a,eual dilution s tseiformed ann I tO ur, u',ed in a y-nse 15 in dun a! PCR u.g& uun' R.x hot th,< waeno m cofflan td 5 n $ a 2X SYRR PCR mastet mix. 0.L a1 ofa 10 uM kainathveittforwatdprinter and U.fl l.gfa 14 uM.kanamvein reverse primer. PCR conditions inchided. an initial incubation 9YC thr.3.0 seConds fOllowed, by 45 of95t fOr 1) iiëcoOds and SrCfor 60 sneonds, A mionn.
Curve. praram. toikwLd the PUt fOrd e"purnose. aeralidating the resu Its. FIG' 42 shows iihc scatter pk of results and in ble 3 shows tiie:Suinwary of the coimt tQr.kanaitelfl, FIG. 4.2 w dPI P re'uk' ott) 0002pv Kanrwc ii R\ \ itn XUR Lut qPCR nagt fis shown n FIG 12, po%rse se Is svc: okeived out of7' te&tion rn10i xokxu'c' pnsent ft 0.0002pg (520 ntokcuies present in 0.Ooipg).
100468] Example 18.: Gen. expresdon.measurenieatiin.ILKerIRNA (004691 The gene pressimi values of tarOts bfvhr4ng bun1anci, wOre measured. ushg stochastic pbcJing. Based on pre ous'assu:mp',tions of transcript abundance', differing eon&.cntitanv uThu n uc total RNA. w u'ed to.est aei of' zeie', (IAPDH, RiM RP Pt. SOFIA. GUSB. TUBB. ABCFI. (i5PD. and TEP The RNA. quantifies used in each reaction \\Oh de',gnea to tatge the tdealenuntm range of L300 noIcuIe\ anti 4ev ucic 0Xi25 p'!.2'5 pg,.i.25 p' 125'g. 12.5 125 ig 2500 p, 650 pg aOd 659 respeet.ivdy. The samoles were added to a.. 12.6.d. reaction containing I ag F.. (oh total RNA I p,l ofa. it) n'4v1 solotiuti cjflNTPs;O.4 iii ofa It) aM speCi& dO timer and 0.4 p! .oi a J.( IIM Pool of 960 41' oligo tae1s. lUbe. rtatdon was incubated at 65°C fcr 5 nhi to denature the RNA and.lben quicld.y'chilkd on ice 4' at of.a SX iiri aud.huflO.r, I of a 0 M Dl F I at otpcasL RNa',e mhThuo (20 unIt I uS (200 an t) ufihe Id pc *i05 \1\U \ C% JSC ii arLscrtpt T d d 0 4 ul nfl ON A cra. C unasi vas adc ed The icacUons;s e iou. bataa at 31 ( fat 60 mm coIIo Ct 3 c' . ks of 9' ( rot 2mm: C tot 2 nm itni 72 C tor 2 tr n tout ni inet1 DNA gkcno v a' added that e eaction was i i.xed. and. moved to a new tube and. i.ticdhated at. 31t Ir 3OmirA. 5 pL. of the reaction vas then added to a 20 ICR reactieti of LXTfaq. Reactimi: hidTei..0I2 iM oC I P 0 05 aM gene'ecdu iouaai d N unci. U 0 aM un ei'4 rovu C p1 une' o.nd 0 aq pnivnwi aw PC ond uots er Q4 fbr n ii to 1kw & d b\ 30 u. o 91 ir 20 T$ m 20 aid 7 °( tnt 20 ec A final nict 1, itt&sn a 7 101 Ulul V,.Ps pc-rfornKd A nosle PC ft \;as puformed unrIg a f'tcd fonsrd pimer and Jrc r&\rrse primer with a Cy3 i.thel at.tacht.d. 0.. otth.ini1&.: PCR wa uscd.as template.Thr the nested. PCR. PCR condidons were the. sanc as fbi. the.first.PCR exccpt:that the 58C step was perlormed at. 55C. itt.. sampl.es.wcre. hybridized to a. detector array at. 37C overnight and cannot t'it fi11os rita usmu. a fluorer*en. reidtr to detoot s Fueb po'tior on JJt4 000Lifled UK C \ dl)tI I he iumher of pontne \)oh td to detci r at. the nt a emicentration ofsampk. Table. 4 s1 ows a summary at the coums fbr {1104701 Thbk.4..
R.PLi.9 20{) GAPDI.I. 376
DHA ____________________
GUSS 19 I'UB3.
pamsjj0cj)___ OP6I' ÷ 30 A.BCFI 3 -k TBP 15 t0047.1i.E.smpie 19: AbMii.utt q tntftatkrn ofniR.NA mO ci)ie d.h-ectiy iysate 1004721 rh example de itho'5 j nethoi to gunue nancupt oount dreoty cet \uICs \ rn use of 40400 eeih trori the Rt mos Rk coil bto washed in PBS were p ccd iii a f4 tube with. : fOht.OWiinL ten 1J ff X-lO(i 5% I pg E Coil total NA, It gi f: oM sokttioaofdNFP\ GAul bf a gene speeitk dU primer and U.4;ai of a lUu* pea: of 960 dT Clilgos. Thi sampks were hctd to 70C for 10 tniiute add chilled no ice t.Ityse 1.06 *ih*c ceils and aik lU). e.priLoer:: Gaune.ai. 4. ofá 5*X first t*aid I tL o.fa (}itv ID rt. It.L1 ofspcns RNe iihihhor (20 UnitS).: II ftL (200 units) oldie vild. ty-e MMLV iC\.Us. .cran t ase and O.A ui...dfTa4DNA. po1vmetae ( nit$ was added. (IIohtr& sampks Were. also petformed lftr the s..me. tcli.nuthhers without the reverse transcniptase. the ete thcubáft-d at 37C fbr 60 miz Uoed h 3 cycles of crc.br 2thin. 55.1 Ica 2 tam and 72 ( tot 2 flhfl mat ot uiaed DN \ elyus tase s'as adned arc toe xcacnoi was. nosed aid t:nove:d.lo a new tubeand incubated at 37 °C tn: 30mm. 5. nIL c-f the reaction was then added to a. 20. iLL.PCR.Irea&inn eomsrstmg of i.X1'arj Reaction. huIfiu*i:02. RM d\ VP U 0 uM ncspxifie orad purnct 00 t\i urnsersl te'erc pum cud 0 1 4L I q pok ne P( R cond tton \ en. 94 tam 2 n ihflon ed b W cy k-or94 C tIn 20 . & i 72°C fur 13 se final inctthauon 2 °C fur 4 1W 1 5% pertormed.A nested IPCR was per.tbrrned usigg a nested fhrward çrimer and the universaL revere primer with. cya label utidehed. 0.l5fd of the jthtia:. PCt& W5i3 aced as template toe the nest-ed PClk. PCR eandidons were same as for the first PCR except that the. $e( ste wps peribnned at 55C. The samp1e were hybridized to a dptec.pr.rrav at I.7C overnight and -caaned the follow n'g eLms using 4 flmmotescence ieder o detect w htb pothons on the arrty uthrd Th&Cy:3 hmbdL. The rmmber ofoosidve spots: wa used to.detet-mIne the. initial concentration of the RPLI 9.transcrjt in the Cells. FIG. 43 shows a dntgram summarizing, the-clai'tatiflr of the stochastic aliii1es:prc co to cells.
[004731 ErnpIe 20 Opthnkathm of eDNA ntheth [00474.! thm eDNA. s othesis reactions werC conducted. The odt:ion.o I thi thre.L jeUoj1s are described below.
1100751 Reaction. l..t Std eniftifti RNA. -m--lOoM d124 --F-Reveme.Trairct.iptasc' [004761 Reaction 2.: Chant cootmi RNA +iOng polv A ranier RNA. + iOnM 2124 + Retse ham srtptM.
1004771 Reacuon I Bead -coutto, BA I I I Øt( 2140 beds Res ar'c Ii anseriptee 1004781 The:eaction:s were incubated ton Ii hour at 42 °C. then diluted to the-. indicated number of. input BAA copies tbr *U cycles of PC.R The PCIR products for each. reaction arc shown.n FiG 112 As shoan in FIG toe RsA eon\enon to LD\-\ u hmghci on heads chmi in.'sr:d.ution..
1004791 flasng no ILL dc'iibed the pte.em n'vcnion.-n-,omc deail be ss of it ustmauon end es4mfl e fur UTThL-of clara-v co unaerstandme ml m I he ohs mou\ to em. of ordinary kiLt in the art that tbe-same can be rcrib'rrned. by modifying or Chani.ng the *i 07 tvSku*itIàaSe ani tr ndiUuns tunüt4bn other $thiSW$ftatit apt*fthntsnta:4tffkrettiboditht&: i:: 1:tó:1 M:*M:S:U, in oi s' :::a:t':, øs ts:.:,ip i::i::S:s a*tin4icaüsce:.ofthckvcFo4kilt S'*hose skjfldin i;.:Mto wh tihiS 3w:_4b.$tGtk meaas IfShSMat pUbLn* * iSiib4b. *4 1 4$iEti*ft *i4ióflè4$ bó Pøó4kS liDs Paragraphs of the invention 1. A digital reverse transcription method comprising: a. contacting a sample comprising a plurality of RNA molecules with a plurality of oligonucleotide tags to produce a labeled-RNA molecule, wherein: i. the plurality of RINA molecules comprise at least 2 mRNA molecules of different sequences; ii. the plurality of oligonucleotide tags comprises at least 2 oligonucleotide tags of different sequences; and iii, the plurality of oligonucleotide tags comprises an oligodT sequence; b. conducting a first strand synthesis reaction by contacting the labeled-RNA molecules with a reverse transcriptase enzyme to produce a labeled-cDNA molecule; and c, detecting the labeled-cDNA molecule by hybridizing the labeled-cDNA molecule to a solid support.
2. The method of paragraph 1, wherein the oligonucleotide tag further comprises a unique identifier region.
3, The method of paragraph 2, wherein the unique identifier region is at least one nucleotide in length.
4. The method of paragraph 1, wherein the oligonucleotide tag further comprises a universal primer binding site.
5. The method of paragraph, wherein the oligonucleotide tag is at least one nucleotide in length.
6. The method of paragraph 1, wherein the solid support is an array.
7. The method of paragraph 1, wherein the solid support is an addressable array.
8. The method of paragraph, wherein the solid support is an Affymetrix 3K tag array, Arrayjet non-contact printed array, or Applied Microarrays Inc (AIVIl) array.
9. The method of paragraph 1, wherein the solid support is a bead.
10. The method of paragraph 1, further comprising conducting a polymerase chain reaction on the Iabeled-cDNA molecule of step (b) to produce a double-stranded labeled-cDNA molecule.
11. The method of paragraph 10, wherein conducting the polymerase chain reaction comprises annealing a first target specific primer to the labeled-cDNA molecule.
12. The method of paragraph 0, wherein conducting the polymerase chain reaction further comprises annealing a universal primer to the universal primer binding site of the oligonucleotide tag.
13. The method of paragraph 10, wherein the polymerase chain reaction comprises absolute PCR, HD-PCR, Next Gen PCR, digital RTA, or any combination thereof.
14. The method of paragraph 10, further comprising conducting a nested PCR reaction on the double-stranded labeled-eDNA molecule.
15. The method of paragraph 14, wherein conducting the nested PUt reaction comprises denaturing the double-stranded labeled cDNA molecule to produce a denatured single-stranded labeled-cDNA molecule.
16. The method of paragraph 14, wherein conducting the nested PCR reaction further comprises annealing a second target specific primer to the denatured single-stranded labeled-cDNA molecule.
17. The method of paragraph 14, wherein conducting the nested PCR reaction further comprises annealing a universal primer to the universal primer binding site of the oligonucleotide tag.
18. The method of paragraph 1, further comprising conducting a sequencing reaction to determine the sequence of at least a portion of the oligonucleotide tag, at least a portion of the labeled-cDNA molecule, a complement thereof; a reverse complement thereof; or any combination thereof.
19. The method of paragraph 1, wherein detecting the labeled-cDNA molecules comprises an array detector, fluorescent reader, non-fluorescent detector, CR reader, or scanner.
20. The method of paragraph 19, wherein the array detector is a flatbed scanner.
21. The method of paragraph 19, wherein the fluorescent reader is a Sensovation, AG fluorescent reader.
22. The method of paragraph 1, wherein detecting the labeled-eDNA molecules comprises detecting the labeled-cDNA molecules hybridized to the solid support.
23. The method of paragraph 1, further comprising conducting a hybridization chain reaction.
24. The method of paragraph 1, wherein the oligonucleotide tag comprises one or more secondary structures.
25. The method of paragraph 24, wherein the one or more secondary structures is a hairpin.
26. The method of paragraph 1, wherein the oligonucleotide tag is linear.
27. A kit comprising: a. a plurality of oligonucleotide tags, wherein the oligonucleotide tag of the plurality of oligonudeotide tags comprises: i. a target specific region; and ii, a unique identifier region; b. an enzyme.
28. The kit of paragraph 27, wherein the enzyme is a reverse transcriptase enzyme.
29. The kit of paragraph 27, wherein the enzyme is a ligase.
30. The kit of paragraph 27, wherein the enzyme is a polymerase.
3] The kit of paragraph 27, wherein the enzyme is an RNase.
32. The kit of paragraph 27, wherein the enzyme is a DNase.
33. The kit of paragraph 27, wherein the enzyme is an endonuclease.
34. The kit of paragraph 27, wherein the oligonucleotide tag is at least 25 nucleotides in length.
35. The kit of paragraph 27, wherein the unique identifier region is at least 10 nucleotides in length.
36. The kit of paragraph 27, wherein the target specific region is at least 10 nucleotides in length.
37. The kit of paragraph 27, wherein the target specific region comprises an oligodT sequence.
38. The kit of paragraph 27, wherein the oligonucleotide tag further comprises a universal primer binding site.
39. The kit of paragraph 27, further comprising a support.
40. The kit of paragraph 39, wherein the support is a semi-solid support, 4]. The kit of paragraph 39, wherein the support is a solid support.
42. The kit of paragraph 41, wherein the solid support is an array.
43. The kit of paragraph 39, wherein the support is an addressable array.
44. The kit of paragraph 39, wherein the support is an Affymetrix 31K tag array, Array,jet non-contact printed array, or Applied Microarrays Inc (AMT) array.
45. The kit of paragraph 39, the support is a bead.
46. The kit of paragraph 27, further comprising a primer.
47. The kit of paragraph 46, wherein the primer is a universal primer.
48. The kit of paragraph 46, wherein the primer binds to the oligonucleotide tag.
49. The kit of paragraph 46, wherein the primer binds to the universal primer binding site of the oligonucleotide tag. 11]
50. The kit of paragraph 27, further comprising a control oligo.
51. The kit of paragraph 50, wherein the control oligo comprises at least 15 nucleotides.
52. The kit of paragraph 50, wherein the control oligo is a bright hybridization control oligo.
53. The kit of paragraph 50, wherein the control oligo is a spike-in template control.
54. The kit of paragraph 27, wherein the oligonucleotide tag further comprises a label.
55. The kit of paragraph 46, wherein the primer further comprises a label.
56. The kit of paragraph 50, wherein the control oligo further comprises a label.
57. The kit of any of paragraphs 54-56, wherein the label is a dye label.
58. The kit of any of paragraphs 54-56, wherein the label is a Cy3 dye.
59. The kit of any of paragraphs 54-56, wherein the label is a Tye563 dye.
60. The kit of paragraph 27, further comprising a buffer.
6]. The kit of paragraph 27, further comprising a carrier, 62. The kit of paragraph 27, further comprising a detergent.
63. The kit of paragraph 27, further comprising a thermal cycler.
64. The kit of paragraph 27, further comprising a sequencer.
65. The kit of paragraph 27, further comprising a hybridization chamber.
66. The kit of paragraph 27, further comprising a detector.
67. The kit of paragraph 27, further comprising an array detector, fluorescent reader, non-fluorescent detector, CR reader, or scanner.
68. The kit of paragraph 67, wherein the fluorescent reader is a Sensovation or AG fluorescent reader.
69. The kit of paragraph 67, wherein the scanner is a flatbed scanner.
70. The kit of paragraph 27, further comprising a computer.
7]. The kit of paragraph 70, wherein the computer comprises a memory device, 72. The kit of paragraph 71, wherein the memory device is capable of storing data.
73. The kit of paragraph 27, further comprising a software program.
74. The kit of paragraph 27, further comprising a computer-readable program.
75. A cell analysis method comprising: a. contacting a sample comprising a plurality of molecules with a plurality of oligonucleotide tags to produce a labeled-molecule, wherein: i. the plurality of molecules comprise at least 2 molecules of different sequences; ii. the p'urality of oligonucleotide tags comprises at least 2 oligonucleotide tags of different sequences; and iii. the sample is from at least one cell; and b, detecting the labeled-molecule by hybridizing the labeled-molecule to a solid support.
76. A clonal amplification method comprising: a. stochastically labeling a plurality of molecules with a plurality of oligonucleotide tags to produce a labeled-molecule, wherein: i. the plurality of molecules comprise at least 2 molecules of different sequences; and ii. the plurality of oligonucleotide tags comprises at least 2 oligonucleotide tags of different sequences; b. amplif'ing the labeled-molecules to produce a labeled-amplicon; and c. detecting the labeled-amplicon.
77. The method of any of paragraphs 75-76, wherein the oligonucleotide tag further comprises a unique identifier region.
78. The method of paragraph 77, wherein the unique identifier region is at least 10 nucleotides in length.
79. The method of paragraph 77, wherein the unique identifier region is cannot hybridize to the molecule.
80. The method of any of paragraphs 75-76, wherein the oligonucleotide tag further comprises a universal primer binding site.
81. The method of any of paragraphs 75-76, wherein the oligonucleotide tag is at least 20 nucleotides in length.
82. The method of any of paragraphs 75-76, wherein the oligonucleotide tag further comprises a target specific region.
83. The method of paragraph 82, wherein the target specific region comprises an oligodT sequence.
84. The method of paragraph 82, wherein the target specific region is at least 10 nucleotides in length.
85. The method of any of paragraphs 75-76, wherein the solid support is an array.
86. The method of any of paragraphs 75-76, wherein the solid support is an addressable array.
87. The method of any of paragraphs 75-76, wherein the solid support is an Affymetrix 3K tag array, Arrayjet non-contact printed array, or Applied Microarrays Inc (AMI) array.
88. The method of any of paragraphs 75-76, wherein the solid support is a bead.
89. The method of any of paragraphs 75-76, further comprising conducting a first strand synthesis reaction to produce a labeled-cDNA molecule.
90. The method of paragraph 75, further comprising conducting a polymerase chain reaction on the labeled-molecule or any product thereof to produce a double-stranded labeled-molecule.
91 The method of paragraph 76, wherein amplifying the labeled-molecule comprises conducting a polymerase chain reaction.
92. The method of any of paragraphs 90-91, wherein conducting the polymerase chain reaction comprises annealing a first target specific primer to the libeled-molecule or any product thereof.
93. The method of any of paragraphs 90-91, wherein conducting the polymerase chain reaction further comprises annealing a universal primer to the universal primer binding site of the oligonucleotide tag.
94, The method of any of paragraphs 90-91, wherein the polymerase chain reaction comprises absolute PCR, I-lD-PCR, Next Gen PCR, digital RTA, or any combination thereof 95. The method of any of paragraphs 75-76, further comprising conducting a nested PCR reaction on the double-stranded labeled-cDNA molecule.
96. The method of paragraph 95, wherein conducting the nested PCR reaction comprises denaturing the labeled-molecule or any product thereof to produce a denatured single-stranded labeled-molecule or any product thereof.
97. The method of any of paragraphs 95-96, wherein conducting the nested PCR reaction further comprises annealing a second target specific primer to the denatured single-stranded labeled-molecule or any product thereof 98. The method of any of paragraphs 95-96, wherein conducting the nested PCR reaction further comprises annealing a universal primer to the universal primer binding site of the oligonucl eoti de tag.
99. The method of any of paragraphs 75-76, further comprising conducting a sequencing reaction to determine the sequence of at least a portion of the oligonucleotide tag, at least a portion of the labeled-molecule, a product thereof, a complement thereof, a reverse complement thereof, or any combination thereof 100. The method of any of paragraphs 75-76, wherein detecting the labeled-molecules or any products thereof comprises an array detector, fluorescent reader, non-fluorescent detector, CR reader, or scanner.
10]. The method of paragraph 00, wherein the fluorescent reader is a Sensovation, or a AG fluorescent reader.
102. The method of paragraph 100, wherein the scanner is a flatbed scanner.
103. The method of any of paragraphs 75-76, wherein detecting the labeled-molecules or any products thereof comprises detecting the labeled-molecules hybridized to the solid support 104. The method of any of paragraphs 75-76, wherein the molecule is a nucleic acid molecule.
105. The method of paragraph 04, wherein the nucleic acid molecule is a DNA molecule.
106. The method of paragraph 104, wherein the nucleic acid molecule is an RNA molecule.
107. The method of any of paragraphs 75-76, wherein the molecule is a peptide.
108. The method of paragraph 107, wherein the peptide is a polypeptide.
109. The method of paragraph 76, wherein the plurality of molecules is from a cell.
110. The method of paragraph 75, wherein the sample is from a single cell.
111, The method of paragraph 75, wherein the sample is from less than about 100 cells.
112. The method of paragraph 75, wherein the sample is from less than about 50 cells.
113. The method of paragraph 75, wherein the sample is from less than about 20 cells.
114. The method of paragraph 75, wherein the sample is from less than about 10 cells.
115. The method of paragraph 75, wherein the sample is from less than about S cells.
116. The method of paragraph 75, wherein the cell is a mammalian cell.
117. The method of paragraph 75, wherein the cell is a human cell.
118. The method of paragraph 75, wherein the cell is from a subject suffering from a disease or condition.
119. The method of paragraph 118, wherein the disease or condition is cancer.
120. The method of paragraph 118, wherein the disease or condition is a pathogenic infection.
121. The method of paragraph 118, wherein the disease or condition is a genetic disorder.
122. The method of any of paragraphs 109-110, wherein the cell is from a healthy subject.
123 The method of any of paragraphs t 09-110, wherein the cell is a diseased cell 124. The method of paragraph 23, wherein the diseased cell is a cancerous cell.
125. The method of any of paragraphs 109-110, wherein the cell is a healthy cell.
126. The method of paragraph 125, wherein the cell is not a diseased or infected cell.
127. The method of any of paragraphs t and 75-76, wherein labeled-molecules are produced by stochastic labeling.
128. A stochastic label-based hybridization chain reaction method, comprising stochastically labeling one or more nucleic acid molecules with a plurality of hairpin oligonucleotide tags, wherein: (a) the hairpin oligonucleotide tag comprises an overhang; and (b) the one or more nucleic acid molecules act as initiators for a hybridization chain reaction.
129. The method of paragraph 128, wherein at least a portion of the hairpin oligonucleotide tag hybridizes to at least a portion of the one or more nucleic acid molecules.
130. The method of paragraph 28, wherein the hairpin oligonucleotide tag comprises an oligodT sequence.
131. The method of paragraph 128, wherein the one or more nucleic acid molecules comprise one or more adapters.
132. The method of paragraph 3, wherein at least a portion of the hairpin oligonucleotide tag hybridizes to at least a portion of the one or more adapters.
133. The method of paragraph 128, wherein at least one hairpin oligonucleotide tag of the plurality of hairpin oligonucleotide tags comprises one or more labels.
134. The method of paragraph 128, wherein at least one hairpin oligonucleotide tag of the plurality of hairpin oligonudeotide tags comprises two or more labels.
135. The method of paragraph 128, wherein each hairpin oligonucleotide tag of the plurality of hairpin oligonucleotide tags comprises one or more labels.
136. The method of paragraph 128, wherein each hairpin oligonucleotide tag of the plurality of hairpin oligonudeotide tags comprises two or more labels.
137. The method of paragraph 128, wherein the hairpin oligonucleotide tag does not comprise a label.
138. The method of paragraph US, wherein the plurality of hairpin oligonucleotide tags comprises one or more hairpin oligonucleotide tags with a 5' overhang, hairpin oligonucleotide tags with a 3' overhang, or a combination thereof 139. The method of paragraph 128, wherein the stem portion of the hairpin oligonucleotide tag is one or more nucleotides in length.
140. The method of paragraph 128, wherein the stem portion of the hairpin oligonucleotide tag is two or more nucleotides in length.
141. The method of paragraph 128, wherein the stem portion of the hairpin oligonucleotide tag is three or more nucleotides in length.
142. The method of paragraph US, wherein the stem portion of the hairpin oligonucleotide tag is four or more nucleotides in length.
143. The method of paragraph 128, wherein the stem portion of the hairpin oligonucleotide tag is five or more nucleotides in length.
144. The method of paragraph 128, wherein the stem portion of the hairpin oligonucleotide tag is six or more nucleotides in length.
145. The method of paragraph 128, wherein the stem portion of the hairpin oligonucleotide tag is seven or more nucleotides in length.
146. The method of paragraph US, wherein the stem portion of the hairpin oligonucleotide tag is eight or more nucleotides in length.
147. The method of paragraph 128, wherein the stem portion of the hairpin oligonucleotide tag is nine or more nucleotides in length.
148. The method of paragraph US, wherein the stem portion of the hairpin oligonucleotide tag is ten or more nueleotides in length.
149. The method of paragraph 128, wherein the loop portion of the hairpin oligonucleotide tag is one or more nucleotides in length.
150, The method of paragraph 128, wherein the loop portion of the hairpin oligonucleotide tag is two or more nucleotides in length.
151. The method of paragraph 128, wherein the loop portion of the hairpin oligonucleotide tag is three or more nucleotides in length.
152. The method of paragraph 128, wherein the loop portion of the hairpin oligonucleotide tag is four or more nucleotides in length.
153. The method of paragraph 128, wherein the loop portion of the hairpin oligonucleotide tag is five or more nucleotides in length.
154. The method of paragraph 128, wherein the loop portion of the hairpin oligonucleotide tag is six or more nucleotides in length.
155. The method of paragraph 128, wherein the loop portion of the hairpin oligonucleotide tag is seven or more nucleotides in length.
156. The method of paragraph 128, wherein the loop portion of the hairpin oligonucleotide tag is eight or more nucleotides in length.
157. The method of paragraph 128, wherein the loop portion of the hairpin oligonucleotide tag is nine or more nucleotides in length.
158. The method of paragraph 128, wherein the loop portion of the hairpin oligonucleotide tag is ten or more nucleotides in length.
159. The method of paragraph 128, wherein the hairpin oligonucleotide tag comprises a unique identifier region.
160. The method of paragraph 159, wherein the unique identifier region is in the ioop portion of the hairpin oligonucleotide tag.
161. The method of paragraph 159, wherein the unique identifier region is in the stem portion of the hairpin oligonucleotide tag.
162. The method of paragraph 159, wherein the unique identifier region is in the overhang portion of the hairpin oligonucleotide tag.
163. The method of paragraph 33, wherein the label comprises a unique identifier region.

Claims (29)

  1. Claims 1. A method for quantifying a nucleic acid molecule in an original sample comprising: a. contacting the original sample comprising a plurality of parent nucleic acid molecules with a plurality of oligonucleotide tags to produce an original labeled sample comprising a plurality of labeled-nucleic acid molecules; b. treating the original labeled sample with a uracil DNA glycosylase (UDG), thereby generating a treated labeled sample; c. amplifying the labeled-nucleic acid molecules from the treated labeled sample to produce an amplified sample comprising a plurality of labeled-amplicons; and d. determining a count of a first parent nucleic acid molecule in the original sample by determining the number of different labeled-amplicons in the amplified sample that originate from the first parent nucleic acid molecule.
  2. 2. The method of claim 1, wherein step (c) comprises producing two or more amplified samples comprising a plurality of labeled-atnplicons.
  3. 3. The method of claim I or claim 2, wherein amplifying the labeled-nucleic acid molecules from the treated labeled sample comprises amplifying the labeled-nucleic acid molecules at least 2, 3, 4,5, 6,7,8,9, or 10 times.
  4. 4. The method of claim 2, wherein producing the two or more amplified samples further comprises amplifying the labeled-amplicons.
  5. 5. The method of claim 1, wherein the plurality of nucleic acid molecules comprise a plurality of RNA molecules.
  6. 6. The method of claim 5, further comprising conducting a first strand synthesis reaction by contacting the plurality of labeled-nucleic acid molecules with a reverse transcriptase enzyme to produce a labeled-cDNA molecule.
  7. 7. The method of claim 6, wherein conducting the first strand synthesis reaction occurs prior to producing the amplified sample.
  8. 8. The method of any one of claims Ito 7, wherein determining the number of different labeled-amplicons in the amplified sample comprises hybridizing the labeled-amplicons to a solid support.
  9. 9. The method of claim I or claim 2, further comprising amplifying the labeled-amplicons from the amplified sample.
  10. 10. The method of claim 9, wherein amplifying the labeled-amplicons from the amplified sample comprises conducting a PCR reaction.
  11. 11. The method of claim 10, wherein the PCR reaction is a nested PCR reaction.
  12. 12. The method of claim 1, wherein the original sample is from one or more cells.
  13. 13. The method of claim 1, wherein an oligonucleotide tag of the plurality of oligonucleotide tags comprises a unique identifier region.
  14. 14. The method of claim 1, wherein an oligonucleotide tag of the plurality of oligonucleotide tags comprises a universal primer binding site.
  15. 15. A clonal amplification method comprising: a. stochastically labeling a plurality of molecules with a plurality of oligonucleotide tags to produce a labeled-molecule, wherein: i. the plurality of molecules comprises at least 2 molecules of different sequences; and ii. the plurality of oligonucleotide tags comprises at least 2 oligonucleotide tags of different sequences; b. amplifying the labeled molecules to produce a labeled-amplicon; c. amplifying the labeled-amplicons by conducting a nested PCR reaction to produce a plurality of nested-PCR amplified labeled-amplicons: and d. detecting the plurality of nested-PCR amplified labeled-amplicons.
  16. 16. The clonal amplification method ofclaiml5, further comprising conducting a first strand synthesis reaction on the labeled-molecule to produce a labeled-eDNA molecule.
  17. 17. The clonal amplification method of claim 15, further comprising conducting a polymerase chain reaction on the labeled-molecule or any product thereof to produce a double-stranded labeled-molecule.
  18. 18. The clonal amplification method of claim 17, wherein conducting the nested PCR reaction comprises denaturing the labeled-amplicon to produce a denatured single-stranded labeled-molecule or any product thereof.
  19. 19. The clonal amplification method of claim 18, wherein conducting the nested PCR reaction further comprises annealing a target specific primer to the denatured single-stranded labeled-molecule or any product thereof.
  20. 20. The clonal amplification method of claim 15, wherein the oligonucleotide tag further comprises a unique identifier region.
  21. 21. The clonal amplification method of claim 20, wherein the unique identifier region is at least 10 nucleotides in length.
  22. 22. The clonal amplification method of claim 20, wherein the unique identifier region cannot hybridize to the molecule.
  23. 23. The clonal amplification method of claim 15, wherein the oligonucleotide tag further comprises a universal primer binding site.
  24. 24. The clonal amplification method of claim 15, wherein the oligonucleotide tag is at least 20 nucleotides in length.
  25. 25. The clonal amplification method of claim 15, wherein the oligonucleotide tag further comprises a target specific region.
  26. 26. The clonal amplification method of claim 25, wherein the target specific region comprises an oligodT sequence.
  27. 27. The clonal amplification method of claim 25, wherein the target specific region is at least 10 nucleotides in length.
  28. 28. The clonal amplification method of claim is, wherein the plurality of molecules is from a cell.
  29. 29. The clonal amplification method of claim 15, wherein amplifying the labeled-molecules to produce a labeled-amplicon comprises amplifying the labeled-nucleic acid molecules at least 2, 3, 4,5,6,7,8,9, or 10 times.
GB1408829.8A 2012-02-27 2013-02-27 A clonal amplification method Active GB2513024B (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US201261603921P 2012-02-27 2012-02-27
US201261745385P 2012-12-21 2012-12-21
GB1319347.9A GB2504240B (en) 2012-02-27 2013-02-27 Compositions and kits for molecular counting of nucleic acids

Publications (3)

Publication Number Publication Date
GB201408829D0 GB201408829D0 (en) 2014-07-02
GB2513024A true GB2513024A (en) 2014-10-15
GB2513024B GB2513024B (en) 2016-08-31

Family

ID=49083250

Family Applications (2)

Application Number Title Priority Date Filing Date
GB1408829.8A Active GB2513024B (en) 2012-02-27 2013-02-27 A clonal amplification method
GB1319347.9A Active GB2504240B (en) 2012-02-27 2013-02-27 Compositions and kits for molecular counting of nucleic acids

Family Applications After (1)

Application Number Title Priority Date Filing Date
GB1319347.9A Active GB2504240B (en) 2012-02-27 2013-02-27 Compositions and kits for molecular counting of nucleic acids

Country Status (11)

Country Link
US (3) US10941396B2 (en)
EP (2) EP2820158B1 (en)
JP (2) JP6375230B2 (en)
CN (1) CN104364392B (en)
AU (2) AU2013226081B2 (en)
CA (2) CA3218231A1 (en)
ES (2) ES2904816T3 (en)
GB (2) GB2513024B (en)
HK (2) HK1194109A1 (en)
SG (1) SG11201405274WA (en)
WO (1) WO2013130674A1 (en)

Families Citing this family (122)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10741034B2 (en) 2006-05-19 2020-08-11 Apdn (B.V.I.) Inc. Security system and method of marking an inventory item and/or person in the vicinity
AU2010232439C1 (en) 2009-04-02 2017-07-13 Fluidigm Corporation Multi-primer amplification method for barcoding of target nucleic acids
US8835358B2 (en) 2009-12-15 2014-09-16 Cellular Research, Inc. Digital counting of individual molecules by stochastic attachment of diverse labels
US9315857B2 (en) 2009-12-15 2016-04-19 Cellular Research, Inc. Digital counting of individual molecules by stochastic attachment of diverse label-tags
SG10201605049QA (en) * 2011-05-20 2016-07-28 Fluidigm Corp Nucleic acid encoding reactions
US9404864B2 (en) 2013-03-13 2016-08-02 Denovo Sciences, Inc. System for imaging captured cells
EP2739587B1 (en) 2011-08-01 2020-05-27 Denovo Sciences Cell capture system
US10466160B2 (en) 2011-08-01 2019-11-05 Celsee Diagnostics, Inc. System and method for retrieving and analyzing particles
JP2014531908A (en) 2011-10-14 2014-12-04 プレジデント アンド フェローズ オブ ハーバード カレッジ Sequencing by structural assembly
EP4108782B1 (en) 2011-12-22 2023-06-07 President and Fellows of Harvard College Compositions and methods for analyte detection
US11021737B2 (en) 2011-12-22 2021-06-01 President And Fellows Of Harvard College Compositions and methods for analyte detection
GB2513024B (en) * 2012-02-27 2016-08-31 Cellular Res Inc A clonal amplification method
US11177020B2 (en) 2012-02-27 2021-11-16 The University Of North Carolina At Chapel Hill Methods and uses for molecular tags
US9914967B2 (en) 2012-06-05 2018-03-13 President And Fellows Of Harvard College Spatial sequencing of nucleic acids using DNA origami probes
US20150011396A1 (en) 2012-07-09 2015-01-08 Benjamin G. Schroeder Methods for creating directional bisulfite-converted nucleic acid libraries for next generation sequencing
US20160040229A1 (en) 2013-08-16 2016-02-11 Guardant Health, Inc. Systems and methods to detect rare mutations and copy number variation
US11913065B2 (en) 2012-09-04 2024-02-27 Guardent Health, Inc. Systems and methods to detect rare mutations and copy number variation
EP2893040B1 (en) 2012-09-04 2019-01-02 Guardant Health, Inc. Methods to detect rare mutations and copy number variation
US10876152B2 (en) 2012-09-04 2020-12-29 Guardant Health, Inc. Systems and methods to detect rare mutations and copy number variation
WO2014108850A2 (en) 2013-01-09 2014-07-17 Yeda Research And Development Co. Ltd. High throughput transcriptome analysis
US9606102B2 (en) 2013-01-26 2017-03-28 Denovo Sciences, Inc. System and method for capturing and analyzing cells
US9963740B2 (en) 2013-03-07 2018-05-08 APDN (B.V.I.), Inc. Method and device for marking articles
US10138509B2 (en) 2013-03-12 2018-11-27 President And Fellows Of Harvard College Method for generating a three-dimensional nucleic acid containing matrix
US9707562B2 (en) 2013-03-13 2017-07-18 Denovo Sciences, Inc. System for capturing and analyzing cells
EP2971130A4 (en) 2013-03-15 2016-10-05 Nugen Technologies Inc Sequential sequencing
US10391490B2 (en) 2013-05-31 2019-08-27 Celsee Diagnostics, Inc. System and method for isolating and analyzing cells
US9856535B2 (en) 2013-05-31 2018-01-02 Denovo Sciences, Inc. System for isolating cells
KR102512979B1 (en) 2013-06-04 2023-03-22 프레지던트 앤드 펠로우즈 오브 하바드 칼리지 Rna-guideded transcriptional regulation
GB2525104B (en) * 2013-08-28 2016-09-28 Cellular Res Inc Massively Parallel Single Cell Nucleic Acid Analysis
EP3058339B1 (en) 2013-10-07 2019-05-22 APDN (B.V.I.) Inc. Multimode image and spectral reader
JP2017504307A (en) 2013-10-07 2017-02-09 セルラー リサーチ， インコーポレイテッド Method and system for digitally counting features on an array
JP6525473B2 (en) 2013-11-13 2019-06-05 ニューゲン テクノロジーズ， インコーポレイテッド Compositions and methods for identifying replicate sequencing leads
JP6571665B2 (en) 2013-12-28 2019-09-04 ガーダント ヘルス， インコーポレイテッド Methods and systems for detecting genetic variants
JP6484636B2 (en) 2014-01-16 2019-03-13 イラミーナ インコーポレーテッド Amplicon preparation and sequencing on solid supports
US20160340720A1 (en) * 2014-02-13 2016-11-24 Cellular Research Methods and compositions for array-based counting
US10745825B2 (en) 2014-03-18 2020-08-18 Apdn (B.V.I.) Inc. Encrypted optical markers for security applications
US10047282B2 (en) 2014-03-18 2018-08-14 Apdn (B.V.I.) Inc. Encrypted optical markers for security applications
US9897597B2 (en) * 2014-04-23 2018-02-20 Children's Medical Center Corporation High-throughput structure determination using nucleic acid calipers
US10179932B2 (en) 2014-07-11 2019-01-15 President And Fellows Of Harvard College Methods for high-throughput labelling and detection of biological features in situ using microscopy
EP3626834B1 (en) * 2014-07-15 2022-09-21 Qiagen Sciences, LLC Semi-random barcodes for nucleic acid analysis
CN107873054B (en) * 2014-09-09 2022-07-12 博德研究所 Droplet-based methods and apparatus for multiplexed single-cell nucleic acid analysis
CN107407638B (en) 2014-12-03 2021-01-12 伊索普莱西斯公司 Analysis and screening of cellular secretion characteristics
EP3259371B1 (en) 2015-02-19 2020-09-02 Becton, Dickinson and Company High-throughput single-cell analysis combining proteomic and genomic information
WO2016138496A1 (en) * 2015-02-27 2016-09-01 Cellular Research, Inc. Spatially addressable molecular barcoding
US11535882B2 (en) 2015-03-30 2022-12-27 Becton, Dickinson And Company Methods and compositions for combinatorial barcoding
WO2016172373A1 (en) 2015-04-23 2016-10-27 Cellular Research, Inc. Methods and compositions for whole transcriptome amplification
US11124823B2 (en) 2015-06-01 2021-09-21 Becton, Dickinson And Company Methods for RNA quantification
US10344336B2 (en) 2015-06-09 2019-07-09 Life Technologies Corporation Methods, systems, compositions, kits, apparatus and computer-readable media for molecular tagging
WO2016210191A1 (en) * 2015-06-23 2016-12-29 Tupac Bio, Inc. Computer-implemented method for designing synthetic dna, and terminal, system and computer-readable medium for the same
EP3347465B1 (en) 2015-09-11 2019-06-26 Cellular Research, Inc. Methods and compositions for nucleic acid library normalization
MX2018005611A (en) 2015-11-03 2018-11-09 Harvard College Method and apparatus for volumetric imaging of a three-dimensional nucleic acid containing matrix.
GB201520341D0 (en) * 2015-11-18 2015-12-30 Randox Lab Ltd And Randox Teoranta Improvements relating to substrates for the attachment of molecules
EP3390658B1 (en) 2015-12-16 2022-08-03 Standard BioTools Inc. High-level multiplex amplification
WO2017106768A1 (en) 2015-12-17 2017-06-22 Guardant Health, Inc. Methods to determine tumor gene copy number by analysis of cell-free dna
JP6685138B2 (en) 2016-01-27 2020-04-22 シスメックス株式会社 Quality control method for nucleic acid amplification, quality control reagent and reagent kit therefor
US11486873B2 (en) 2016-03-31 2022-11-01 Ontera Inc. Multipore determination of fractional abundance of polynucleotide sequences in a sample
CN109070130B (en) 2016-04-11 2022-03-22 亚普蒂恩（B V I）公司 Method for marking cellulose products
CN116200465A (en) * 2016-04-25 2023-06-02 哈佛学院董事及会员团体 Hybrid chain reaction method for in situ molecular detection
EP3452614B1 (en) 2016-05-02 2023-06-28 Becton, Dickinson and Company Accurate molecular barcoding
US10301677B2 (en) 2016-05-25 2019-05-28 Cellular Research, Inc. Normalization of nucleic acid libraries
WO2017205691A1 (en) * 2016-05-26 2017-11-30 Cellular Research, Inc. Molecular label counting adjustment methods
US10640763B2 (en) 2016-05-31 2020-05-05 Cellular Research, Inc. Molecular indexing of internal sequences
US10202641B2 (en) 2016-05-31 2019-02-12 Cellular Research, Inc. Error correction in amplification of samples
KR101915701B1 (en) * 2016-06-16 2018-11-07 한국한의학연구원 Method for measuring mutation rate
CN109923216B (en) 2016-08-31 2024-08-02 哈佛学院董事及会员团体 Method of combining detection of biomolecules into a single assay using fluorescence in situ sequencing
CN109983125B (en) 2016-08-31 2024-06-04 哈佛学院董事及会员团体 Method for generating a library of nucleic acid sequences for detection by fluorescence in situ sequencing
CA3034924A1 (en) 2016-09-26 2018-03-29 Cellular Research, Inc. Measurement of protein expression using reagents with barcoded oligonucleotide sequences
US10460828B2 (en) 2016-09-30 2019-10-29 Lifeos Genomics Corporation Method of nucleic acid fragment detection
CN106498040B (en) * 2016-10-12 2018-10-23 浙江大学 A kind of molecular labeling microballon and the unicellular sequencing approach of high throughput based on the molecular labeling microballon
US10995371B2 (en) 2016-10-13 2021-05-04 Apdn (B.V.I.) Inc. Composition and method of DNA marking elastomeric material
CA3021676C (en) 2016-10-24 2021-03-09 Two Pore Guys, Inc. Fractional abundance of polynucleotide sequences in a sample
KR20190077061A (en) 2016-11-08 2019-07-02 셀룰러 리서치, 인크. Cell labeling method
CN109952612B (en) 2016-11-08 2023-12-01 贝克顿迪金森公司 Method for classifying expression profiles
EP3559231B1 (en) 2016-11-11 2022-08-17 Bio-Rad Laboratories, Inc. Methods for processing nucleic acid samples
EP3538671A1 (en) * 2016-11-11 2019-09-18 Roche Innovation Center Copenhagen A/S Therapeutic oligonucleotides capture and detection
WO2018089910A2 (en) 2016-11-11 2018-05-17 IsoPlexis Corporation Compositions and methods for the simultaneous genomic, transcriptomic and proteomic analysis of single cells
WO2018098372A1 (en) 2016-11-22 2018-05-31 IsoPlexis Corporation Systems, devices and methods for cell capture and methods of manufacture thereof
EP3513346B1 (en) * 2016-11-30 2020-04-15 Microsoft Technology Licensing, LLC Dna random access storage system via ligation
US20180179577A1 (en) * 2016-12-22 2018-06-28 Jiaming HU Lipid-polymer Hybrid Nanoparticle Biochip and Application Thereof
WO2018132610A1 (en) 2017-01-13 2018-07-19 Cellular Research, Inc. Hydrophilic coating of fluidic channels
US11319583B2 (en) 2017-02-01 2022-05-03 Becton, Dickinson And Company Selective amplification using blocking oligonucleotides
US11085071B2 (en) * 2017-02-01 2021-08-10 Spot Biosystems Ltd. Highly stable and specific molecular beacons encapsulated in cationic lipoplex nanoparticles and application thereof
US10920274B2 (en) 2017-02-21 2021-02-16 Apdn (B.V.I.) Inc. Nucleic acid coated submicron particles for authentication
JP6983906B2 (en) * 2017-04-11 2021-12-17 ニューゲン テクノロジーズ， インコーポレイテッド Quantitative and qualitative library
US20180291443A1 (en) * 2017-04-11 2018-10-11 Nugen Technologies, Inc. Library Quantitation And Qualification
WO2018191672A1 (en) 2017-04-14 2018-10-18 President And Fellows Of Harvard College Methods for generation of cell-derived microfilament network
AU2018281745B2 (en) 2017-06-05 2022-05-19 Becton, Dickinson And Company Sample indexing for single cells
JP2019024453A (en) * 2017-08-02 2019-02-21 株式会社リコー Rna concentration quantitative method, rna concentration quantitative device, and rna concentration quantitative apparatus
US10391493B2 (en) 2017-08-29 2019-08-27 Celsee Diagnostics, Inc. System and method for isolating and analyzing cells
CN107447055A (en) * 2017-09-19 2017-12-08 中国动物疫病预防控制中心 Pig parvoviral droplet digital pcr absolute quantitation detection kit
US11099202B2 (en) 2017-10-20 2021-08-24 Tecan Genomics, Inc. Reagent delivery system
WO2019126209A1 (en) 2017-12-19 2019-06-27 Cellular Research, Inc. Particles associated with oligonucleotides
EP3543351B1 (en) 2018-03-19 2022-08-10 Ricoh Company, Ltd. Nucleic acid sample-contained container, method for producing nucleic acid sample-contained container, and nucleic acid sample
WO2019213237A1 (en) 2018-05-03 2019-11-07 Becton, Dickinson And Company Molecular barcoding on opposite transcript ends
US11773441B2 (en) 2018-05-03 2023-10-03 Becton, Dickinson And Company High throughput multiomics sample analysis
SG11202101934SA (en) 2018-07-30 2021-03-30 Readcoor Llc Methods and systems for sample processing or analysis
US11473133B2 (en) 2018-09-24 2022-10-18 Second Genome, Inc. Methods for validation of microbiome sequence processing and differential abundance analyses via multiple bespoke spike-in mixtures
WO2020072380A1 (en) 2018-10-01 2020-04-09 Cellular Research, Inc. Determining 5' transcript sequences
US11932849B2 (en) 2018-11-08 2024-03-19 Becton, Dickinson And Company Whole transcriptome analysis of single cells using random priming
CN113195717A (en) 2018-12-13 2021-07-30 贝克顿迪金森公司 Selective extension in single cell whole transcriptome analysis
US11371076B2 (en) 2019-01-16 2022-06-28 Becton, Dickinson And Company Polymerase chain reaction normalization through primer titration
ES2945227T3 (en) 2019-01-23 2023-06-29 Becton Dickinson Co Antibody Associated Oligonucleotides
US12071617B2 (en) 2019-02-14 2024-08-27 Becton, Dickinson And Company Hybrid targeted and whole transcriptome amplification
EP3935185A1 (en) * 2019-03-04 2022-01-12 King Abdullah University Of Science And Technology Compositions and methods of labeling nucleic acids and sequencing and analysis thereof
US10633693B1 (en) 2019-04-16 2020-04-28 Celsee Diagnostics, Inc. System and method for leakage control in a particle capture system
US11965208B2 (en) 2019-04-19 2024-04-23 Becton, Dickinson And Company Methods of associating phenotypical data and single cell sequencing data
CN114072490A (en) 2019-05-07 2022-02-18 伯乐实验室有限公司 System and method for automated single cell processing
US11273439B2 (en) 2019-05-07 2022-03-15 Bio-Rad Laboratories, Inc. System and method for target material retrieval from microwells
CA3143241A1 (en) 2019-06-14 2020-12-17 Bio-Rad Laboratories, Inc. System and method for automated single cell processing and analyses
CA3147297A1 (en) * 2019-07-16 2021-01-21 Meliolabs Inc. Methods and devices for single-cell based digital high resolution melt
CN114051534A (en) 2019-07-22 2022-02-15 贝克顿迪金森公司 Single cell chromatin immunoprecipitation sequencing assay
KR20220097404A (en) 2019-10-10 2022-07-07 1859, 인크. Methods and systems for microfluidic screening
JP7522189B2 (en) 2019-11-08 2024-07-24 ベクトン・ディキンソン・アンド・カンパニー Use of Random Priming to Obtain Full-Length V(D)J Information for Immune Repertoire Sequencing
EP4090763A1 (en) 2020-01-13 2022-11-23 Becton Dickinson and Company Methods and compositions for quantitation of proteins and rna
CN113275053A (en) 2020-02-03 2021-08-20 帝肯基因组学公司 Reagent storage system
US11504719B2 (en) 2020-03-12 2022-11-22 Bio-Rad Laboratories, Inc. System and method for receiving and delivering a fluid for sample processing
EP4150118A1 (en) 2020-05-14 2023-03-22 Becton Dickinson and Company Primers for immune repertoire profiling
US11932901B2 (en) 2020-07-13 2024-03-19 Becton, Dickinson And Company Target enrichment using nucleic acid probes for scRNAseq
CN114096678A (en) * 2020-07-31 2022-02-25 北京寻因生物科技有限公司 Multiple nucleic acid co-labeling support, and preparation method and application thereof
KR20230088443A (en) 2020-10-19 2023-06-19 바이오 래드 래버러토리스 인코오포레이티드 Systems and methods for rapid multiplexed sample processing with applications for nucleic acid amplification assays
EP4247967A1 (en) 2020-11-20 2023-09-27 Becton, Dickinson and Company Profiling of highly expressed and lowly expressed proteins
CN113337585A (en) * 2021-07-23 2021-09-03 苏州英泽生物医药科技有限公司 Small RNA quantitative detection method

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050019776A1 (en) * 2002-06-28 2005-01-27 Callow Matthew James Universal selective genome amplification and universal genotyping system
US20080299609A1 (en) * 2007-03-12 2008-12-04 Sungkyunkwan University Foundation For Corporate Collaboration Uracil-DNA glycosylase of psychrobacter sp. HJ147 and use thereof
US20110294689A1 (en) * 2010-05-27 2011-12-01 Affymetrix, Inc Multiplex Amplification Methods

Family Cites Families (611)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4437975A (en) 1977-07-20 1984-03-20 Mobil Oil Corporation Manufacture of lube base stock oil
US4510244A (en) 1980-04-17 1985-04-09 The Board Of Trustees Of The Leland Stanford Jr. University Cell labeling with antigen-coupled microspheres
US5242794A (en) 1984-12-13 1993-09-07 Applied Biosystems, Inc. Detection of specific sequences in nucleic acids
US4725536A (en) 1985-09-19 1988-02-16 Genetics Institute, Inc. Reagent polynucleotide complex with multiple target binding regions, and kit and methods
US6150517A (en) 1986-11-24 2000-11-21 Gen-Probe Methods for making oligonucleotide probes for the detection and/or quantitation of non-viral organisms
IL86724A (en) 1987-06-19 1995-01-24 Siska Diagnostics Inc Method and kits for the amplification and detection of nucleic acid sequences
IE72468B1 (en) 1987-07-31 1997-04-09 Univ Leland Stanford Junior Selective amplification of target polynucleotide sequences
US5149625A (en) 1987-08-11 1992-09-22 President And Fellows Of Harvard College Multiplex analysis of DNA
US5124246A (en) 1987-10-15 1992-06-23 Chiron Corporation Nucleic acid multimers and amplified nucleic acid hybridization assays using same
US5656731A (en) 1987-10-15 1997-08-12 Chiron Corporation Nucleic acid-amplified immunoassay probes
JP2650159B2 (en) 1988-02-24 1997-09-03 アクゾ・ノベル・エヌ・ベー Nucleic acid amplification method
CA1340807C (en) 1988-02-24 1999-11-02 Lawrence T. Malek Nucleic acid amplification process
US4988617A (en) 1988-03-25 1991-01-29 California Institute Of Technology Method of detecting a nucleotide change in nucleic acids
EP0426756A4 (en) 1988-07-26 1991-11-21 Genelabs Incorporated Rna and dna amplification techniques
KR0148265B1 (en) 1988-12-16 1998-10-15 에프.지이.엠 헤르만스 Self-sustained sequence replication system
US5856092A (en) 1989-02-13 1999-01-05 Geneco Pty Ltd Detection of a nucleic acid sequence or a change therein
US6551784B2 (en) 1989-06-07 2003-04-22 Affymetrix Inc Method of comparing nucleic acid sequences
US6040138A (en) 1995-09-15 2000-03-21 Affymetrix, Inc. Expression monitoring by hybridization to high density oligonucleotide arrays
US5424186A (en) 1989-06-07 1995-06-13 Affymax Technologies N.V. Very large scale immobilized polymer synthesis
US6346413B1 (en) 1989-06-07 2002-02-12 Affymetrix, Inc. Polymer arrays
US5800992A (en) 1989-06-07 1998-09-01 Fodor; Stephen P.A. Method of detecting nucleic acids
US5744101A (en) 1989-06-07 1998-04-28 Affymax Technologies N.V. Photolabile nucleoside protecting groups
US5925525A (en) 1989-06-07 1999-07-20 Affymetrix, Inc. Method of identifying nucleotide differences
US5547839A (en) 1989-06-07 1996-08-20 Affymax Technologies N.V. Sequencing of surface immobilized polymers utilizing microflourescence detection
US6309822B1 (en) 1989-06-07 2001-10-30 Affymetrix, Inc. Method for comparing copy number of nucleic acid sequences
US5527681A (en) 1989-06-07 1996-06-18 Affymax Technologies N.V. Immobilized molecular synthesis of systematically substituted compounds
US5871928A (en) 1989-06-07 1999-02-16 Fodor; Stephen P. A. Methods for nucleic acid analysis
US5242974A (en) 1991-11-22 1993-09-07 Affymax Technologies N.V. Polymer reversal on solid surfaces
US5143854A (en) 1989-06-07 1992-09-01 Affymax Technologies N.V. Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereof
US5252743A (en) 1989-11-13 1993-10-12 Affymax Technologies N.V. Spatially-addressable immobilization of anti-ligands on surfaces
US5200314A (en) 1990-03-23 1993-04-06 Chiron Corporation Polynucleotide capture assay employing in vitro amplification
US5494810A (en) 1990-05-03 1996-02-27 Cornell Research Foundation, Inc. Thermostable ligase-mediated DNA amplifications system for the detection of genetic disease
CA2090015A1 (en) 1990-08-24 1992-02-25 Brant J. Bassam Dna amplification fingerprinting
JP3164814B2 (en) 1990-08-27 2001-05-14 科学技術振興事業団 Preparation of homogenized cDNA library
WO1992007095A1 (en) 1990-10-15 1992-04-30 Stratagene Arbitrarily primed polymerase chain reaction method for fingerprinting genomes
DE69132843T2 (en) 1990-12-06 2002-09-12 Affymetrix, Inc. (N.D.Ges.D.Staates Delaware) Identification of nucleic acids in samples
JPH04337446A (en) 1991-05-15 1992-11-25 Hitachi Ltd Method and device for measuring fine grain and constant quantity method
US5981179A (en) 1991-11-14 1999-11-09 Digene Diagnostics, Inc. Continuous amplification reaction
US5412087A (en) 1992-04-24 1995-05-02 Affymax Technologies N.V. Spatially-addressable immobilization of oligonucleotides and other biological polymers on surfaces
US5550215A (en) 1991-11-22 1996-08-27 Holmes; Christopher P. Polymer reversal on solid surfaces
US5324633A (en) 1991-11-22 1994-06-28 Affymax Technologies N.V. Method and apparatus for measuring binding affinity
US5677195A (en) 1991-11-22 1997-10-14 Affymax Technologies N.V. Combinatorial strategies for polymer synthesis
US5384261A (en) 1991-11-22 1995-01-24 Affymax Technologies N.V. Very large scale immobilized polymer synthesis using mechanically directed flow paths
US5424413A (en) 1992-01-22 1995-06-13 Gen-Probe Incorporated Branched nucleic acid probes
US5573905A (en) 1992-03-30 1996-11-12 The Scripps Research Institute Encoded combinatorial chemical libraries
US5541061A (en) 1992-04-29 1996-07-30 Affymax Technologies N.V. Methods for screening factorial chemical libraries
US5981176A (en) 1992-06-17 1999-11-09 City Of Hope Method of detecting and discriminating between nucleic acid sequences
US5491074A (en) 1993-04-01 1996-02-13 Affymax Technologies Nv Association peptides
JP3954092B2 (en) 1993-06-25 2007-08-08 アフィメトリックス インコーポレイテッド Nucleic acid sequence hybridization and sequencing
US5837832A (en) 1993-06-25 1998-11-17 Affymetrix, Inc. Arrays of nucleic acid probes on biological chips
US5858659A (en) 1995-11-29 1999-01-12 Affymetrix, Inc. Polymorphism detection
US6087186A (en) 1993-07-16 2000-07-11 Irori Methods and apparatus for synthesizing labeled combinatorial chemistry libraries
US5500356A (en) 1993-08-10 1996-03-19 Life Technologies, Inc. Method of nucleic acid sequence selection
US6045996A (en) 1993-10-26 2000-04-04 Affymetrix, Inc. Hybridization assays on oligonucleotide arrays
US6309823B1 (en) 1993-10-26 2001-10-30 Affymetrix, Inc. Arrays of nucleic acid probes for analyzing biotransformation genes and methods of using the same
US5681697A (en) 1993-12-08 1997-10-28 Chiron Corporation Solution phase nucleic acid sandwich assays having reduced background noise and kits therefor
US5631734A (en) 1994-02-10 1997-05-20 Affymetrix, Inc. Method and apparatus for detection of fluorescently labeled materials
US5578832A (en) 1994-09-02 1996-11-26 Affymetrix, Inc. Method and apparatus for imaging a sample on a device
US6090555A (en) 1997-12-11 2000-07-18 Affymetrix, Inc. Scanned image alignment systems and methods
US5714330A (en) 1994-04-04 1998-02-03 Lynx Therapeutics, Inc. DNA sequencing by stepwise ligation and cleavage
DE69503126T2 (en) 1994-05-05 1998-11-12 Beckman Instruments Inc REPETITIVE OLIGONUCLEOTIDE MATRIX
US5571639A (en) 1994-05-24 1996-11-05 Affymax Technologies N.V. Computer-aided engineering system for design of sequence arrays and lithographic masks
US6495518B1 (en) 1994-06-13 2002-12-17 Vanderbilt University Method for importing biologically active molecules into cells
GB2293238A (en) 1994-09-13 1996-03-20 Inceltec Ltd Primers for replication and/or amplification reactions
US5710000A (en) 1994-09-16 1998-01-20 Affymetrix, Inc. Capturing sequences adjacent to Type-IIs restriction sites for genomic library mapping
US5846719A (en) 1994-10-13 1998-12-08 Lynx Therapeutics, Inc. Oligonucleotide tags for sorting and identification
US5695934A (en) 1994-10-13 1997-12-09 Lynx Therapeutics, Inc. Massively parallel sequencing of sorted polynucleotides
US5604097A (en) 1994-10-13 1997-02-18 Spectragen, Inc. Methods for sorting polynucleotides using oligonucleotide tags
US6013445A (en) 1996-06-06 2000-01-11 Lynx Therapeutics, Inc. Massively parallel signature sequencing by ligation of encoded adaptors
US6600996B2 (en) 1994-10-21 2003-07-29 Affymetrix, Inc. Computer-aided techniques for analyzing biological sequences
US5795716A (en) 1994-10-21 1998-08-18 Chee; Mark S. Computer-aided visualization and analysis system for sequence evaluation
EP0709466B1 (en) 1994-10-28 2006-09-27 Gen-Probe Incorporated Compositions and methods for the simultaneous detection and quantification of multiple specific nucleic acid sequences
US5599695A (en) 1995-02-27 1997-02-04 Affymetrix, Inc. Printing molecular library arrays using deprotection agents solely in the vapor phase
US5959098A (en) 1996-04-17 1999-09-28 Affymetrix, Inc. Substrate preparation process
KR19990008052A (en) 1995-04-25 1999-01-25 마이클 피 노바 Remotely programmable matrix with storage device and uses thereof
US5624711A (en) 1995-04-27 1997-04-29 Affymax Technologies, N.V. Derivatization of solid supports and methods for oligomer synthesis
US5648245A (en) 1995-05-09 1997-07-15 Carnegie Institution Of Washington Method for constructing an oligonucleotide concatamer library by rolling circle replication
US5968740A (en) 1995-07-24 1999-10-19 Affymetrix, Inc. Method of Identifying a Base in a Nucleic Acid
US5733729A (en) 1995-09-14 1998-03-31 Affymetrix, Inc. Computer-aided probability base calling for arrays of nucleic acid probes on chips
US5763175A (en) 1995-11-17 1998-06-09 Lynx Therapeutics, Inc. Simultaneous sequencing of tagged polynucleotides
US5854033A (en) 1995-11-21 1998-12-29 Yale University Rolling circle replication reporter systems
US6300063B1 (en) 1995-11-29 2001-10-09 Affymetrix, Inc. Polymorphism detection
US6147205A (en) 1995-12-15 2000-11-14 Affymetrix, Inc. Photocleavable protecting groups and methods for their use
US6613544B1 (en) 1995-12-22 2003-09-02 Amgen Inc. Osteoprotegerin
US5962271A (en) 1996-01-03 1999-10-05 Cloutech Laboratories, Inc. Methods and compositions for generating full-length cDNA having arbitrary nucleotide sequence at the 3'-end
US5830712A (en) 1996-02-06 1998-11-03 Allelix Biopharmaceuticals Inc. Selective template deletion method
US6458530B1 (en) 1996-04-04 2002-10-01 Affymetrix Inc. Selecting tag nucleic acids
WO1997043611A1 (en) 1996-05-16 1997-11-20 Affymetrix, Inc. Systems and methods for detection of labeled materials
EP1736554B1 (en) 1996-05-29 2013-10-09 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions
JPH1021373A (en) 1996-07-05 1998-01-23 Fuji Photo Film Co Ltd Picture analyzing device
US7169556B2 (en) 1996-07-29 2007-01-30 Nanosphere, Inc. Nanoparticles having oligonucleotides attached thereto and uses therefor
US20020172953A1 (en) 1996-07-29 2002-11-21 Mirkin Chad A. Movement of biomolecule-coated nanoparticles in an electric field
US6984491B2 (en) 1996-07-29 2006-01-10 Nanosphere, Inc. Nanoparticles having oligonucleotides attached thereto and uses therefor
US6100029A (en) 1996-08-14 2000-08-08 Exact Laboratories, Inc. Methods for the detection of chromosomal aberrations
US5935793A (en) 1996-09-27 1999-08-10 The Chinese University Of Hong Kong Parallel polynucleotide sequencing method using tagged primers
US6124092A (en) 1996-10-04 2000-09-26 The Perkin-Elmer Corporation Multiplex polynucleotide capture methods and compositions
US6117631A (en) 1996-10-29 2000-09-12 Polyprobe, Inc. Detection of antigens via oligonucleotide antibody conjugates
US6046005A (en) 1997-01-15 2000-04-04 Incyte Pharmaceuticals, Inc. Nucleic acid sequencing with solid phase capturable terminators comprising a cleavable linking group
CA2291180A1 (en) 1997-05-23 1998-11-26 Lynx Therapeutics, Inc. System and apparatus for sequential processing of analytes
ATE319856T1 (en) 1997-06-13 2006-03-15 Affymetrix Inc A Delaware Corp METHOD FOR DETECTING GENE POLYMORPHISMS AND ALLELE EXPRESSION USING PROBE CHIPS
US6333179B1 (en) 1997-06-20 2001-12-25 Affymetrix, Inc. Methods and compositions for multiplex amplification of nucleic acids
US6974669B2 (en) 2000-03-28 2005-12-13 Nanosphere, Inc. Bio-barcodes based on oligonucleotide-modified nanoparticles
US6420108B2 (en) 1998-02-09 2002-07-16 Affymetrix, Inc. Computer-aided display for comparative gene expression
ATE264523T1 (en) 1997-07-25 2004-04-15 Affymetrix Inc A Delaware Corp METHOD FOR CREATING A BIO-INFORMATICS DATABASE
JP2001514907A (en) 1997-08-15 2001-09-18 アフィメトリックス・インコーポレーテッド Polymorph detection using cluster analysis
US6399334B1 (en) 1997-09-24 2002-06-04 Invitrogen Corporation Normalized nucleic acid libraries and methods of production thereof
DE69829402T2 (en) 1997-10-31 2006-04-13 Affymetrix, Inc. (a Delaware Corp.), Santa Clara EXPRESSION PROFILES IN ADULTS AND FOLDS ORGANS
US6013449A (en) 1997-11-26 2000-01-11 The United States Of America As Represented By The Department Of Health And Human Services Probe-based analysis of heterozygous mutations using two-color labelling
WO1999028505A1 (en) 1997-12-03 1999-06-10 Curagen Corporation Methods and devices for measuring differential gene expression
US6265163B1 (en) 1998-01-09 2001-07-24 Lynx Therapeutics, Inc. Solid phase selection of differentially expressed genes
US6428752B1 (en) 1998-05-14 2002-08-06 Affymetrix, Inc. Cleaning deposit devices that form microarrays and the like
US6269846B1 (en) 1998-01-13 2001-08-07 Genetic Microsystems, Inc. Depositing fluid specimens on substrates, resulting ordered arrays, techniques for deposition of arrays
US6201639B1 (en) 1998-03-20 2001-03-13 James W. Overbeck Wide field of view and high speed scanning microscopy
US6185030B1 (en) 1998-03-20 2001-02-06 James W. Overbeck Wide field of view and high speed scanning microscopy
US6020135A (en) 1998-03-27 2000-02-01 Affymetrix, Inc. P53-regulated genes
US5936324A (en) 1998-03-30 1999-08-10 Genetic Microsystems Inc. Moving magnet scanner
US6787308B2 (en) 1998-07-30 2004-09-07 Solexa Ltd. Arrayed biomolecules and their use in sequencing
WO2000014282A1 (en) 1998-09-04 2000-03-16 Lynx Therapeutics, Inc. Method of screening for genetic polymorphism
US6185561B1 (en) 1998-09-17 2001-02-06 Affymetrix, Inc. Method and apparatus for providing and expression data mining database
US6262216B1 (en) 1998-10-13 2001-07-17 Affymetrix, Inc. Functionalized silicon compounds and methods for their synthesis and use
DE69929542T2 (en) 1998-10-27 2006-09-14 Affymetrix, Inc., Santa Clara COMPLEXITY MANAGEMENT AND ANALYSIS OF GENOMIC DNA
US6480791B1 (en) 1998-10-28 2002-11-12 Michael P. Strathmann Parallel methods for genomic analysis
US6197554B1 (en) 1998-11-20 2001-03-06 Shi-Lung Lin Method for generating full-length cDNA library from single cells
US6436675B1 (en) 1999-09-28 2002-08-20 Maxygen, Inc. Use of codon-varied oligonucleotide synthesis for synthetic shuffling
US20020019005A1 (en) 1999-02-18 2002-02-14 Arcaris, Inc. Process for identification of genes encoding proteins having cell proliferation-promoting activity
US6177248B1 (en) 1999-02-24 2001-01-23 Affymetrix, Inc. Downstream genes of tumor suppressor WT1
US6629040B1 (en) 1999-03-19 2003-09-30 University Of Washington Isotope distribution encoded tags for protein identification
JP2002539849A (en) 1999-03-26 2002-11-26 ホワイトヘッド インスチチュート フォアー バイオメディカル リサーチ Universal array
US6355431B1 (en) 1999-04-20 2002-03-12 Illumina, Inc. Detection of nucleic acid amplification reactions using bead arrays
EP1046717B1 (en) 1999-04-20 2010-10-06 National Institute of Advanced Industrial Science and Technology Method and probes for determining a concentration of target nucleic acid molecules and method for analyzing data obtained by the method
US6218803B1 (en) 1999-06-04 2001-04-17 Genetic Microsystems, Inc. Position sensing with variable capacitance transducers
US6372813B1 (en) 1999-06-25 2002-04-16 Motorola Methods and compositions for attachment of biomolecules to solid supports, hydrogels, and hydrogel arrays
US6326148B1 (en) 1999-07-12 2001-12-04 The Regents Of The University Of California Detection of copy number changes in colon cancer
US6440706B1 (en) 1999-08-02 2002-08-27 Johns Hopkins University Digital amplification
JP2001078768A (en) * 1999-09-16 2001-03-27 Otsuka Pharmaceut Co Ltd Assay of gene expression
SE9903988D0 (en) 1999-11-03 1999-11-03 Amersham Pharm Biotech Ab Method of analysis
US6582938B1 (en) 2001-05-11 2003-06-24 Affymetrix, Inc. Amplification of nucleic acids
US6489114B2 (en) 1999-12-17 2002-12-03 Bio Merieux Process for labeling a ribonucleic acid, and labeled RNA fragments which are obtained thereby
CN1500150A (en) 1999-12-29 2004-05-26 ��Ĭ���� Methods for amplifying and detecting multiple polynucleotides on solid phase support
CA2396810A1 (en) 2000-01-24 2001-07-26 Phylos, Inc. Sensitive, multiplexed diagnostic assays for protein analysis
US7022479B2 (en) 2000-01-24 2006-04-04 Compound Therapeutics, Inc. Sensitive, multiplexed diagnostic assays for protein analysis
US20030097222A1 (en) 2000-01-25 2003-05-22 Craford David M. Method, system, and computer software for providing a genomic web portal
US6235483B1 (en) * 2000-01-31 2001-05-22 Agilent Technologies, Inc. Methods and kits for indirect labeling of nucleic acids
US20050214825A1 (en) 2000-02-07 2005-09-29 John Stuelpnagel Multiplex sample analysis on universal arrays
DK1259643T3 (en) 2000-02-07 2009-02-23 Illumina Inc Method for Detecting Nucleic Acid Using Universal Priming
GB2364054B (en) 2000-03-24 2002-05-29 Smithkline Beecham Corp Method of amplifying quinolone-resistance-determining-regions and identifying polymorphic variants thereof
DK1715063T3 (en) 2000-03-29 2011-05-16 Lgc Ltd Hydration beacon and method for rapid sequence detection and distinction
DK2278029T3 (en) 2000-04-10 2016-10-03 Taxon Biosciences Inc Methods of study and genetic analysis of populations
US20030207300A1 (en) 2000-04-28 2003-11-06 Matray Tracy J. Multiplex analytical platform using molecular tags
US7630063B2 (en) 2000-08-02 2009-12-08 Honeywell International Inc. Miniaturized cytometer for detecting multiple species in a sample
US6511809B2 (en) 2000-06-13 2003-01-28 E. I. Du Pont De Nemours And Company Method for the detection of an analyte by means of a nucleic acid reporter
AU2001282881B2 (en) 2000-07-07 2007-06-14 Visigen Biotechnologies, Inc. Real-time sequence determination
CA2418502A1 (en) 2000-08-11 2002-03-07 Nanosphere Inc. Nanoparticles having oligonucleotides attached thereto and uses therefor
CA2356123A1 (en) 2000-08-25 2002-02-25 Riken Method of preparing normalized and/or subtracted cdna
US6934408B2 (en) 2000-08-25 2005-08-23 Amnis Corporation Method and apparatus for reading reporter labeled beads
ATE380883T1 (en) 2000-10-24 2007-12-15 Univ Leland Stanford Junior DIRECT MULTIPLEX CHARACTERIZATION OF GENOMIC DNA
WO2002046472A2 (en) 2000-12-08 2002-06-13 Nanosphere, Inc. Nanoparticles having oligonucleotides attached thereto and uses therefor
US20020142345A1 (en) 2000-12-22 2002-10-03 Nelsen Anita J. Methods for encoding and decoding complex mixtures in arrayed assays
US20030049616A1 (en) 2001-01-08 2003-03-13 Sydney Brenner Enzymatic synthesis of oligonucleotide tags
WO2002070684A2 (en) 2001-01-11 2002-09-12 Lion Bioscience Ag Gene library for screening methods
US6988040B2 (en) 2001-01-11 2006-01-17 Affymetrix, Inc. System, method, and computer software for genotyping analysis and identification of allelic imbalance
US20020183936A1 (en) 2001-01-24 2002-12-05 Affymetrix, Inc. Method, system, and computer software for providing a genomic web portal
CA2435612C (en) 2001-01-25 2014-12-23 Tm Bioscience Corporation Polynucleotides for use as tags and tag complements, manufacture and use thereof
US20030120432A1 (en) 2001-01-29 2003-06-26 Affymetrix, Inc. Method, system and computer software for online ordering of custom probe arrays
KR20030082535A (en) 2001-03-09 2003-10-22 뉴젠 테크놀로지스 인코포레이티드 Methods and compositions for amplification of rna sequences
US6804679B2 (en) 2001-03-12 2004-10-12 Affymetrix, Inc. System, method, and user interfaces for managing genomic data
JP2005233974A (en) 2001-03-21 2005-09-02 Olympus Corp Biochemical inspection method
US7027932B2 (en) 2001-03-21 2006-04-11 Olympus Optical Co., Ltd. Biochemical examination method
WO2002079490A2 (en) 2001-03-28 2002-10-10 Nanosphere, Inc. Bio-barcodes based on oligonucleotide-modified particles
US20030170675A1 (en) 2001-04-11 2003-09-11 The Gov't Of The U.S Of America As Represented By The Secretary Of The Dept. Of Health & Human Serv. Methods of manipulating nucleic acids
WO2002083922A2 (en) 2001-04-11 2002-10-24 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Modified random primers for probe labeling
AUPR480901A0 (en) 2001-05-04 2001-05-31 Genomics Research Partners Pty Ltd Diagnostic method for assessing a condition of a performance animal
CA2344599C (en) 2001-05-07 2011-07-12 Bioneer Corporation Selective polymerase chain reaction of dna of which base sequence is completely unknown
US6905827B2 (en) 2001-06-08 2005-06-14 Expression Diagnostics, Inc. Methods and compositions for diagnosing or monitoring auto immune and chronic inflammatory diseases
WO2002101358A2 (en) 2001-06-11 2002-12-19 Illumina, Inc. Multiplexed detection methods
JP4244534B2 (en) 2001-06-12 2009-03-25 横河電機株式会社 Biochip
US7473767B2 (en) 2001-07-03 2009-01-06 The Institute For Systems Biology Methods for detection and quantification of analytes in complex mixtures
US6830931B2 (en) 2001-07-12 2004-12-14 Automated Cell, Inc. Method and apparatus for monitoring of proteins and cells
US20030100995A1 (en) 2001-07-16 2003-05-29 Affymetrix, Inc. Method, system and computer software for variant information via a web portal
US6872529B2 (en) 2001-07-25 2005-03-29 Affymetrix, Inc. Complexity management of genomic DNA
EP1478774A4 (en) 2001-10-09 2006-11-29 Nanosphere Inc Nanoparticles having oligonucleotides attached thereto and uses therefor
AU2002360272A1 (en) 2001-10-10 2003-04-22 Superarray Bioscience Corporation Detecting targets by unique identifier nucleotide tags
US20030077611A1 (en) 2001-10-24 2003-04-24 Sention Methods and systems for dynamic gene expression profiling
WO2003050242A2 (en) 2001-11-13 2003-06-19 Rubicon Genomics Inc. Dna amplification and sequencing using dna molecules generated by random fragmentation
EP2196544A1 (en) 2001-11-21 2010-06-16 Applied Biosystems, LLC Kit for ligation detection assays using codeable labels
US20030175749A1 (en) 2001-12-08 2003-09-18 Jong-Yoon Chun Annealing control primer and its uses
US20040002818A1 (en) 2001-12-21 2004-01-01 Affymetrix, Inc. Method, system and computer software for providing microarray probe data
US20030120431A1 (en) 2001-12-21 2003-06-26 Affymetrix, Inc. Method and computer software product for genomic alignment and assessment of the transcriptome
US8110351B2 (en) 2002-01-16 2012-02-07 Invitrogen Dynal As Method for isolating nucleic acids and protein from a single sample
JP2005515792A (en) * 2002-01-29 2005-06-02 グローバル ジェノミクス アクティエボラーグ Methods and means for manipulating nucleic acids
CA2474509C (en) 2002-02-14 2012-01-31 Immunivest Corporation Methods and algorithms for cell enumeration in a low-cost cytometer
US7689022B2 (en) 2002-03-15 2010-03-30 Affymetrix, Inc. System, method, and product for scanning of biological materials
US20030186251A1 (en) 2002-04-01 2003-10-02 Brookhaven Science Associates, Llc Genome sequence tags
US20050175993A1 (en) 2002-04-12 2005-08-11 Chia-Lin Wei Method for making full-length coding sequence cDNA libraries
US20040126840A1 (en) 2002-12-23 2004-07-01 Affymetrix, Inc. Method, system and computer software for providing genomic ontological data
US20040049354A1 (en) 2002-04-26 2004-03-11 Affymetrix, Inc. Method, system and computer software providing a genomic web portal for functional analysis of alternative splice variants
US6808906B2 (en) 2002-05-08 2004-10-26 Rigel Pharmaceuticals, Inc. Directionally cloned random cDNA expression vector libraries, compositions and methods of use
US20070178478A1 (en) 2002-05-08 2007-08-02 Dhallan Ravinder S Methods for detection of genetic disorders
EP1362929A3 (en) 2002-05-17 2004-05-19 Affymetrix, Inc. Methods for genotyping
GB0212391D0 (en) 2002-05-29 2002-07-10 Axis Shield Asa Assay
US20040096368A1 (en) 2002-06-28 2004-05-20 Igen International, Inc. Assay systems and components
US7192700B2 (en) 2002-12-20 2007-03-20 Orchid Cellmark Inc. Methods and compositions for conducting primer extension and polymorphism detection reactions
WO2004018623A2 (en) 2002-08-16 2004-03-04 Clinical Microarrays, Inc. Substrates for isolating, reacting and microscopically analyzing materials
CA2497420C (en) 2002-09-03 2016-04-19 Quanta Biosciences, Inc. Improved compositions and methods for cdna synthesis
US7361821B2 (en) 2002-09-20 2008-04-22 Intel Corporation Controlled alignment of nanobarcodes encoding specific information for scanning probe microscopy (SPM) reading
EP1546376B9 (en) 2002-09-30 2009-08-12 F.Hoffmann-La Roche Ag Oligonucleotides for genotyping thymidylate synthase gene
WO2004040001A2 (en) 2002-10-02 2004-05-13 California Institute Of Technology Microfluidic nucleic acid analysis
CA2505472A1 (en) 2002-11-11 2004-05-27 Affymetrix, Inc. Methods for identifying dna copy number changes
US7822555B2 (en) 2002-11-11 2010-10-26 Affymetrix, Inc. Methods for identifying DNA copy number changes
US7704687B2 (en) 2002-11-15 2010-04-27 The Johns Hopkins University Digital karyotyping
US20040224325A1 (en) 2002-12-20 2004-11-11 Caliper Life Sciences, Inc. Single molecule amplification and detection of DNA
JP2006520463A (en) 2003-01-23 2006-09-07 ユー．エス． ジェノミクス， インコーポレイテッド Methods for analyzing polymer populations
US7575865B2 (en) 2003-01-29 2009-08-18 454 Life Sciences Corporation Methods of amplifying and sequencing nucleic acids
DE602004029560D1 (en) 2003-03-07 2010-11-25 Rubicon Genomics Inc AMPLIFICATION AND ANALYSIS OF TOTAL GENOME AND TOTAL TRANSCRIPTIBLE LIBRARIES PRODUCED BY A DNA POLYMERIZATION PROCEDURE
US8206913B1 (en) 2003-03-07 2012-06-26 Rubicon Genomics, Inc. Amplification and analysis of whole genome and whole transcriptome libraries generated by a DNA polymerization process
US7269518B2 (en) 2003-04-30 2007-09-11 Agilent Technologies, Inc. Chemical array reading
US20040259118A1 (en) 2003-06-23 2004-12-23 Macevicz Stephen C. Methods and compositions for nucleic acid sequence analysis
US20050042654A1 (en) 2003-06-27 2005-02-24 Affymetrix, Inc. Genotyping methods
EP1641809B2 (en) 2003-07-05 2018-10-03 The Johns Hopkins University Method and compositions for detection and enumeration of genetic variations
EP1651776A2 (en) 2003-07-24 2006-05-03 Qiagen GmbH Method for the reverse transcription and/or amplification of nucleic acids
US7317415B2 (en) 2003-08-08 2008-01-08 Affymetrix, Inc. System, method, and product for scanning of biological materials employing dual analog integrators
JP2007502116A (en) 2003-08-13 2007-02-08 アフィメトリックス インコーポレイテッド Methods and kits for preparing nucleic acid samples
GB0319332D0 (en) 2003-08-16 2003-09-17 Astrazeneca Ab Amplification
US20050048498A1 (en) 2003-08-29 2005-03-03 Applera Corporation Compositions, methods, and kits for assembling probes
US7341837B2 (en) 2003-09-02 2008-03-11 Lawton Robert L Soluble analyte detection and amplification
US7354706B2 (en) 2003-09-09 2008-04-08 The Regents Of The University Of Colorado, A Body Corporate Use of photopolymerization for amplification and detection of a molecular recognition event
CA2536565A1 (en) 2003-09-10 2005-05-12 Althea Technologies, Inc. Expression profiling using microarrays
JP2007512811A (en) 2003-11-10 2007-05-24 インベスチゲン， インコーポレイテッド Methods for preparing nucleic acids for detection
EP1709203A2 (en) 2004-01-23 2006-10-11 Lingvitae AS Improving polynucleotide ligation reactions
ATE451478T1 (en) 2004-02-12 2009-12-15 Population Genetics Technologi GENETIC ANALYSIS USING SEQUENCE-SPECIFIC SORTING
US20100216153A1 (en) 2004-02-27 2010-08-26 Helicos Biosciences Corporation Methods for detecting fetal nucleic acids and diagnosing fetal abnormalities
US7432055B2 (en) 2004-03-05 2008-10-07 Uchicago Argonne Llc Dual phase multiplex polymerase chain reaction
US7294466B2 (en) 2004-05-07 2007-11-13 Cepheid Multiplexed detection of biological agents
US20050250147A1 (en) 2004-05-10 2005-11-10 Macevicz Stephen C Digital profiling of polynucleotide populations
JP2008505345A (en) 2004-06-07 2008-02-21 アイアールエム エルエルシー Dispensing system, software, and related methods
US20060035258A1 (en) 2004-08-06 2006-02-16 Affymetrix, Inc. Methods for identifying DNA copy number changes
US20060041385A1 (en) 2004-08-18 2006-02-23 Bauer Kenneth D Method of quantitating proteins and genes in cells using a combination of immunohistochemistry and in situ hybridization
US7713697B2 (en) 2004-08-27 2010-05-11 Gen-Probe Incorporated Methods and kits for amplifying DNA
CN101056883B (en) 2004-09-10 2012-10-10 人类遗传标记控股有限公司 Amplification blocker for intercalating nucleic acid (ina) containing intercalating pseudo nucleic acid (IPN)
EP1647600A3 (en) 2004-09-17 2006-06-28 Affymetrix, Inc. (A US Entity) Methods for identifying biological samples by addition of nucleic acid bar-code tags
US7435084B2 (en) 2004-12-14 2008-10-14 Align Technology, Inc. System and methods for casting physical tooth model
KR20070105967A (en) 2004-11-03 2007-10-31 아이리스 몰레큘라 다이아그노스틱스, 인코오포레이티드 Homogeneous analyte detection
EP1836302B1 (en) * 2004-12-23 2010-04-14 Amersham Biosciences Corp. Ligation-based rna amplification
EP2418018B1 (en) 2004-12-23 2013-05-22 Abbott Point of Care Inc. Methods for the separation nucleic acids
US9222129B2 (en) 2005-02-10 2015-12-29 Riken Method for amplification of nucleotide sequence
US7407757B2 (en) 2005-02-10 2008-08-05 Population Genetics Technologies Genetic analysis by sequence-specific sorting
US7393665B2 (en) 2005-02-10 2008-07-01 Population Genetics Technologies Ltd Methods and compositions for tagging and identifying polynucleotides
EP1856293A2 (en) 2005-03-16 2007-11-21 Compass Genetics, Llc Methods and compositions for assay readouts on multiple analytical platforms
EP2518161A1 (en) 2005-03-18 2012-10-31 Fluidigm Corporation Method for detection of mutant alleles
US20090264635A1 (en) 2005-03-25 2009-10-22 Applera Corporation Methods and compositions for depleting abundant rna transcripts
US20060263789A1 (en) 2005-05-19 2006-11-23 Robert Kincaid Unique identifiers for indicating properties associated with entities to which they are attached, and methods for using
US7695886B2 (en) 2005-05-19 2010-04-13 Fuji Xerox Co., Ltd. Process for producing resin particle liquid dispersion for electrostatic image developing toner, electrostatic image developing toner and production process thereof
US8407013B2 (en) 2005-06-07 2013-03-26 Peter K. Rogan AB initio generation of single copy genomic probes
US20070065844A1 (en) 2005-06-08 2007-03-22 Massachusetts Institute Of Technology Solution-based methods for RNA expression profiling
US7796815B2 (en) 2005-06-10 2010-09-14 The Cleveland Clinic Foundation Image analysis of biological objects
US20070020640A1 (en) 2005-07-21 2007-01-25 Mccloskey Megan L Molecular encoding of nucleic acid templates for PCR and other forms of sequence analysis
US8486621B2 (en) 2005-08-11 2013-07-16 Cornell Research Foundation, Inc. Nucleic acid-based matrixes
EP2485052B1 (en) 2005-09-13 2015-05-06 Affymetrix, Inc. Encoded microparticles
US20070117121A1 (en) 2005-09-16 2007-05-24 Hutchison Stephen K cDNA library preparation
US8831887B2 (en) 2005-10-12 2014-09-09 The Research Foundation For The State University Of New York Absolute PCR quantification
US8313711B2 (en) 2005-11-01 2012-11-20 Freeslate, Inc. Liquid dispensing for high-throughput experimentation
US7375211B2 (en) * 2005-11-18 2008-05-20 Kou Zhong C Method for detection and quantification of T-cell receptor Vβ repertoire
US20080070303A1 (en) 2005-11-21 2008-03-20 West Michael D Methods to accelerate the isolation of novel cell strains from pluripotent stem cells and cells obtained thereby
US7636465B2 (en) 2005-12-09 2009-12-22 Cytyc Corporation Cross-frame object reconstruction for image-based cytology applications
US7544473B2 (en) 2006-01-23 2009-06-09 Population Genetics Technologies Ltd. Nucleic acid analysis using sequence tokens
US7537897B2 (en) * 2006-01-23 2009-05-26 Population Genetics Technologies, Ltd. Molecular counting
US8492098B2 (en) 2006-02-21 2013-07-23 The Trustees Of Tufts College Methods and arrays for target analyte detection and determination of reaction components that affect a reaction
US20080038727A1 (en) * 2006-03-10 2008-02-14 Applera Corporation MicroRNA and Messenger RNA Detection on Arrays
US7880142B2 (en) 2006-04-04 2011-02-01 Sidec Technologies Ab Extended electron tomography
US20080194414A1 (en) 2006-04-24 2008-08-14 Albert Thomas J Enrichment and sequence analysis of genomic regions
US9074242B2 (en) 2010-02-12 2015-07-07 Raindance Technologies, Inc. Digital analyte analysis
WO2007136874A2 (en) 2006-05-18 2007-11-29 President And Fellows Of Harvard College Genomic library construction
US7833716B2 (en) 2006-06-06 2010-11-16 Gen-Probe Incorporated Tagged oligonucleotides and their use in nucleic acid amplification methods
WO2007147079A2 (en) 2006-06-14 2007-12-21 Living Microsystems, Inc. Rare cell analysis using sample splitting and dna tags
WO2008021123A1 (en) 2006-08-07 2008-02-21 President And Fellows Of Harvard College Fluorocarbon emulsion stabilizing surfactants
US8586006B2 (en) 2006-08-09 2013-11-19 Institute For Systems Biology Organ-specific proteins and methods of their use
EP2069472A4 (en) 2006-10-05 2012-06-20 Nanopoint Inc Systems and methods for active microfluidic cell handling
WO2008057163A2 (en) 2006-10-09 2008-05-15 Carnegie Mellon University Preparation of functional gel particles with a dual crosslink network
WO2008047428A1 (en) 2006-10-18 2008-04-24 Osaki Electric Co., Ltd. Electronic watthour meter
WO2008051928A2 (en) 2006-10-23 2008-05-02 The Salk Institute For Biological Studies Target-oriented whole genome amplification of nucliec acids
US8603749B2 (en) 2006-11-15 2013-12-10 Biospherex, LLC Multitag sequencing ecogenomics analysis-US
US8050476B2 (en) 2006-11-22 2011-11-01 General Electric Company Heart rate demodulation of periodic movement in ultrasound data for object identification
EP2677308B1 (en) 2006-12-14 2017-04-26 Life Technologies Corporation Method for fabricating large scale FET arrays
JP5489469B2 (en) 2007-01-16 2014-05-14 オリンパス株式会社 Fluorescence signal analysis apparatus and fluorescence signal analysis method
US9063133B2 (en) 2007-01-30 2015-06-23 The Regents Of The University Of California Methods and devices for biomolecular arrays
US20080269068A1 (en) 2007-02-06 2008-10-30 President And Fellows Of Harvard College Multiplex decoding of sequence tags in barcodes
WO2008096318A2 (en) 2007-02-09 2008-08-14 Koninklijke Philips Electronics N.V. Identification system
US8003312B2 (en) 2007-02-16 2011-08-23 The Board Of Trustees Of The Leland Stanford Junior University Multiplex cellular assays using detectable cell barcodes
SI2472264T1 (en) 2007-02-27 2016-06-30 SentoClone International AB Karolinska Institute Science Park Multiplex detection of tumour cells using a panel of agents binding to extracellular markers
WO2008109176A2 (en) 2007-03-07 2008-09-12 President And Fellows Of Harvard College Assays and other reactions involving droplets
JP2008256428A (en) 2007-04-03 2008-10-23 Intec Systems Institute Inc Method for detecting block position of microarray image
US20080268508A1 (en) 2007-04-30 2008-10-30 Sowlay Mohankumar R Methods and kits for negative selection of desired nucleic acid sequences
US20080274458A1 (en) 2007-05-01 2008-11-06 Latham Gary J Nucleic acid quantitation methods
US20090061513A1 (en) 2007-05-15 2009-03-05 Picovitro Ab Cell sorting and cell cultivation methods
CN101720359A (en) 2007-06-01 2010-06-02 454生命科学公司 System and meth0d for identification of individual samples from a multiplex mixture
US8883691B2 (en) 2007-06-25 2014-11-11 Affymetrix, Inc. Encoded microparticles
US7635566B2 (en) 2007-06-29 2009-12-22 Population Genetics Technologies Ltd. Methods and compositions for isolating nucleic acid sequence variants
DK2036989T3 (en) 2007-09-12 2012-10-22 Pasteur Institut Polynucleotide Suitable for Single Cell Based Reporter Assay for Monitoring Gene Expression Patterns with High Spatiotemporal Resolution
EP2203749B1 (en) 2007-10-05 2012-08-29 Affymetrix, Inc. Highly multiplexed particle-based assays
US10300482B2 (en) 2010-12-09 2019-05-28 Akonni Biosystems, Inc. Sample analysis system
US20090253586A1 (en) 2008-02-21 2009-10-08 Gentel Biosciences, Inc. Substrates for multiplexed assays and uses thereof
US8687189B2 (en) 2008-03-03 2014-04-01 Ajjer, Llc Analysis of arrays by laser induced breakdown spectroscopy
US8206925B2 (en) 2008-04-14 2012-06-26 Medical Diagnostic Laboratories, Llc Splice variants of human IL-23 receptor (IL-23R) mRNA and use of a delta 9 isoform in predicting inflammatory bowel diseases
DE102008025656B4 (en) * 2008-05-28 2016-07-28 Genxpro Gmbh Method for the quantitative analysis of nucleic acids, markers therefor and their use
US20090298709A1 (en) 2008-05-28 2009-12-03 Affymetrix, Inc. Assays for determining telomere length and repeated sequence copy number
US20100120097A1 (en) 2008-05-30 2010-05-13 Board Of Regents, The University Of Texas System Methods and compositions for nucleic acid sequencing
US20100069250A1 (en) 2008-08-16 2010-03-18 The Board Of Trustees Of The Leland Stanford Junior University Digital PCR Calibration for High Throughput Sequencing
US8476013B2 (en) 2008-09-16 2013-07-02 Sequenom, Inc. Processes and compositions for methylation-based acid enrichment of fetal nucleic acid from a maternal sample useful for non-invasive prenatal diagnoses
US8312249B1 (en) 2008-10-10 2012-11-13 Apple Inc. Dynamic trampoline and structured code generation in a signed code environment
US9080211B2 (en) 2008-10-24 2015-07-14 Epicentre Technologies Corporation Transposon end compositions and methods for modifying nucleic acids
WO2010053980A2 (en) 2008-11-04 2010-05-14 The Johns Hopkins University Dna integrity assay (dia) for cancer diagnostics, using confocal fluorescence spectroscopy
CA2744003C (en) 2008-11-21 2016-03-15 Mediomics Llc Biosensor for detecting multiple epitopes on a target
US20100159533A1 (en) 2008-11-24 2010-06-24 Helicos Biosciences Corporation Simplified sample preparation for rna analysis
US8492096B2 (en) 2008-12-24 2013-07-23 Boris Pasche TGFBR1 expression modifies risk for colorectal cancer
AU2010206843B2 (en) * 2009-01-20 2015-01-29 The Board Of Trustees Of The Leland Stanford Junior University Single cell gene expression for diagnosis, prognosis and identification of drug targets
US20100323348A1 (en) 2009-01-31 2010-12-23 The Regents Of The University Of Colorado, A Body Corporate Methods and Compositions for Using Error-Detecting and/or Error-Correcting Barcodes in Nucleic Acid Amplification Process
WO2010093872A2 (en) * 2009-02-13 2010-08-19 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Molecular-based method of cancer diagnosis and prognosis
CA2754446A1 (en) 2009-03-05 2010-09-10 Daiichi Sankyo Company, Limited Pyridine derivative
EP2230312A1 (en) 2009-03-19 2010-09-22 Helmholtz-Zentrum für Infektionsforschung GmbH Probe compound for detecting and isolating enzymes and means and methods using the same
WO2010117620A2 (en) 2009-03-30 2010-10-14 Illumina, Inc. Gene expression analysis in single cells
AU2010232439C1 (en) 2009-04-02 2017-07-13 Fluidigm Corporation Multi-primer amplification method for barcoding of target nucleic acids
WO2010127186A1 (en) 2009-04-30 2010-11-04 Prognosys Biosciences, Inc. Nucleic acid constructs and methods of use
FR2945545B1 (en) 2009-05-14 2011-08-05 Univ Aix Marseille Ii METHOD FOR DETECTION OF PROCARYOTE DNA EXTRACTED FROM A SAMPLE SAMPLE
US20120058520A1 (en) 2009-05-14 2012-03-08 Wako Pure Chemical Industries, Ltd. Method for synthesis of double-stranded dna corresponding to rna, and method for amplification of the dna
US8673627B2 (en) 2009-05-29 2014-03-18 Life Technologies Corporation Apparatus and methods for performing electrochemical reactions
GB0909923D0 (en) 2009-06-09 2009-07-22 Oxford Gene Tech Ip Ltd Picowell capture devices for analysing single cells or other particles
US20120058902A1 (en) 2009-06-25 2012-03-08 Livingston Robert J Method of measuring adaptive immunity
CN102686728B (en) * 2009-06-29 2018-09-18 卢米耐克斯公司 Chimeric primers with hairpin conformation and its application method
US8330957B2 (en) 2009-06-29 2012-12-11 Hager Enviromental and Atmospheric Technologies, LLC Device and method for quantification of gases in plumes by remote sensing
CN102482668A (en) 2009-08-20 2012-05-30 群体遗传学科技有限公司 Compositions and methods for intramolecular nucleic acid rearrangement
WO2011028818A2 (en) 2009-09-01 2011-03-10 Trustees Of Boston University High throughput multichannel reader and uses thereof
EP2473625B1 (en) 2009-09-01 2018-03-07 Koninklijke Philips N.V. Devices and methods for microarray selection
US9625454B2 (en) 2009-09-04 2017-04-18 The Research Foundation For The State University Of New York Rapid and continuous analyte processing in droplet microfluidic devices
WO2011041308A1 (en) 2009-09-30 2011-04-07 Quest Diagnostics Investments Incorporated Bcr-abl truncation mutations
US9315857B2 (en) * 2009-12-15 2016-04-19 Cellular Research, Inc. Digital counting of individual molecules by stochastic attachment of diverse label-tags
US8835358B2 (en) 2009-12-15 2014-09-16 Cellular Research, Inc. Digital counting of individual molecules by stochastic attachment of diverse labels
AU2011207544A1 (en) 2010-01-19 2012-09-06 Verinata Health, Inc. Identification of polymorphic sequences in mixtures of genomic DNA by whole genome sequencing
EP2848704B1 (en) 2010-01-19 2018-08-29 Verinata Health, Inc Sequencing methods for prenatal diagnoses
EP2529026B1 (en) 2010-01-25 2013-11-13 Rd Biosciences Inc. Self-folding amplification of target nucleic acid
JP5901046B2 (en) 2010-02-19 2016-04-06 国立大学法人 千葉大学 Novel alternative splicing variant of OATP1B3 mRNA
WO2011106738A2 (en) 2010-02-25 2011-09-01 Fred Hutchinson Cancer Research Center Use of tcr clonotypes as biomarkers for disease
US8951940B2 (en) 2010-04-01 2015-02-10 Illumina, Inc. Solid-phase clonal amplification and related methods
AU2011237729B2 (en) 2010-04-05 2014-04-03 Prognosys Biosciences, Inc. Spatially encoded biological assays
SG185544A1 (en) 2010-05-14 2012-12-28 Fluidigm Corp Nucleic acid isolation methods
EP2580351B1 (en) 2010-06-09 2018-08-29 Keygene N.V. Combinatorial sequence barcodes for high throughput screening
US20120004132A1 (en) 2010-07-02 2012-01-05 Affymetrix, Inc. Detection of Nucleic Acids and Proteins
WO2012004834A1 (en) 2010-07-07 2012-01-12 株式会社アドバンテスト Testing device and testing method
EP2407242A1 (en) 2010-07-13 2012-01-18 Dublin City University Direct clone analysis and selection technology
US20120040843A1 (en) 2010-08-12 2012-02-16 Dublin City University Centrifugal capture system
EP2623613B8 (en) 2010-09-21 2016-09-07 Population Genetics Technologies Ltd. Increasing confidence of allele calls with molecular counting
EP2436766A1 (en) 2010-09-29 2012-04-04 Deutsches Krebsforschungszentrum Means and methods for improved protein interaction screening
WO2012042374A2 (en) 2010-10-01 2012-04-05 Anssi Jussi Nikolai Taipale Method of determining number or concentration of molecules
US20120088691A1 (en) 2010-10-08 2012-04-12 Gao Chen Highly multiplexed real-time pcr using encoded microbeads
WO2012048341A1 (en) 2010-10-08 2012-04-12 President And Fellows Of Harvard College High-throughput single cell barcoding
US20130225623A1 (en) 2010-10-27 2013-08-29 Mount Sinai School Of Medicine Methods of Treating Psychiatric or Neurological Disorders with MGLUR Antagonists
AU2011336707A1 (en) 2010-11-29 2013-07-04 Dako Denmark A/S Methods and systems for analyzing images of specimens processed by a programmable quantitative assay
US9394511B2 (en) 2010-12-05 2016-07-19 Wenbin Jiang Rapid single cell based parallel biological cell sorter
ES2615733T3 (en) 2010-12-16 2017-06-08 Gigagen, Inc. Methods for parallel mass analysis of nucleic acids in individual cells
JP6328934B2 (en) * 2010-12-22 2018-05-23 ナテラ， インコーポレイテッド Noninvasive prenatal testing
WO2012092515A2 (en) 2010-12-30 2012-07-05 Life Technologies Corporation Methods, systems, and computer readable media for nucleic acid sequencing
EP3567121B1 (en) 2011-01-17 2023-08-30 Life Technologies Corporation Workflow for detection of ligands using nucleic acids
WO2012103154A1 (en) 2011-01-24 2012-08-02 Nugen Technologies, Inc. Stem-loop composite rna-dna adaptor-primers: compositions and methods for library generation, amplification and other downstream manipulations
US20120190021A1 (en) 2011-01-25 2012-07-26 Aria Diagnostics, Inc. Detection of genetic abnormalities
WO2012106385A2 (en) 2011-01-31 2012-08-09 Apprise Bio, Inc. Methods of identifying multiple epitopes in cells
WO2012108864A1 (en) 2011-02-08 2012-08-16 Illumina, Inc. Selective enrichment of nucleic acids
US9150852B2 (en) 2011-02-18 2015-10-06 Raindance Technologies, Inc. Compositions and methods for molecular labeling
SG192870A1 (en) 2011-02-23 2013-09-30 Regents Board Of Use of template switching for dna synthesis
WO2012129363A2 (en) 2011-03-24 2012-09-27 President And Fellows Of Harvard College Single cell nucleic acid detection and analysis
KR20120110431A (en) 2011-03-29 2012-10-10 에스케이하이닉스 주식회사 A semiconductor memory apparatus
GB201106254D0 (en) 2011-04-13 2011-05-25 Frisen Jonas Method and product
DK3246416T3 (en) 2011-04-15 2024-09-02 Univ Johns Hopkins SECURE SEQUENCE SYSTEM
WO2012149042A2 (en) 2011-04-25 2012-11-01 Bio-Rad Laboratories, Inc. Methods and compositions for nucleic acid analysis
US8946389B2 (en) 2011-04-25 2015-02-03 University Of Washington Compositions and methods for multiplex biomarker profiling
DK3415619T3 (en) 2011-04-28 2021-03-08 Univ Leland Stanford Junior IDENTIFICATION OF POLYNUCLEOTIDS ASSOCIATED WITH A SAMPLE
GB201108259D0 (en) 2011-05-17 2011-06-29 Cambridge Entpr Ltd Gel beads in microfluidic droplets
SG10201605049QA (en) 2011-05-20 2016-07-28 Fluidigm Corp Nucleic acid encoding reactions
HUE062102T2 (en) 2011-05-24 2023-09-28 BioNTech SE Individualized vaccines for cancer
WO2012162621A1 (en) 2011-05-26 2012-11-29 Brandeis University Methods for suppression pcr
US8841071B2 (en) 2011-06-02 2014-09-23 Raindance Technologies, Inc. Sample multiplexing
US9752176B2 (en) 2011-06-15 2017-09-05 Ginkgo Bioworks, Inc. Methods for preparative in vitro cloning
WO2012177639A2 (en) 2011-06-22 2012-12-27 Alnylam Pharmaceuticals, Inc. Bioprocessing and bioproduction using avian cell lines
WO2013003498A2 (en) 2011-06-27 2013-01-03 Life Technologies Corporation Acoustic cytometry methods and protocols
WO2013019075A2 (en) 2011-08-01 2013-02-07 연세대학교산학협력단 Method of preparing nucleic acid molecules
WO2013022961A1 (en) 2011-08-08 2013-02-14 3The Broad Institute Compositions and methods for co-amplifying subsequences of a nucleic acid fragment sequence
JP5816486B2 (en) 2011-08-18 2015-11-18 オリンパス株式会社 Fluorescence observation apparatus, fluorescence observation system, and fluorescence image processing method of fluorescence observation apparatus
EP3263718A1 (en) 2011-09-16 2018-01-03 Lexogen GmbH Nucleic acid transcription method
WO2013062931A2 (en) 2011-10-24 2013-05-02 Furchak Jennifer A molecular beacon based assay for the detection of biomarkers of breast cancer metastasis
US9777338B2 (en) 2011-11-11 2017-10-03 Meso Scale Technologies, Llc. Co-binder assisted assay methods
KR101337094B1 (en) 2011-11-30 2013-12-05 삼성에스디에스 주식회사 Apparatus and method for sequence alignment
WO2013096802A1 (en) 2011-12-22 2013-06-27 Ibis Biosciences, Inc. Amplification primers and methods
WO2013117595A2 (en) 2012-02-07 2013-08-15 Illumina Cambridge Limited Targeted enrichment and amplification of nucleic acids on a support
EP2823303A4 (en) 2012-02-10 2015-09-30 Raindance Technologies Inc Molecular diagnostic screening assay
WO2013123125A1 (en) 2012-02-17 2013-08-22 President And Fellows Of Harvard College Assembly of nucleic acid sequences in emulsions
EP3309262B1 (en) 2012-02-24 2019-09-25 Bio-Rad Laboratories, Inc. Labeling and sample preparation for sequencing
US11177020B2 (en) 2012-02-27 2021-11-16 The University Of North Carolina At Chapel Hill Methods and uses for molecular tags
GB2513024B (en) * 2012-02-27 2016-08-31 Cellular Res Inc A clonal amplification method
WO2013137737A1 (en) 2012-03-16 2013-09-19 Flexgen B.V. Methods of creating and screening of dna encoded combinatorial libraries of chemical antibodies
US9803239B2 (en) 2012-03-29 2017-10-31 Complete Genomics, Inc. Flow cells for high density array chips
EP2647426A1 (en) 2012-04-03 2013-10-09 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Replication of distributed nucleic acid molecules with preservation of their relative distribution through hybridization-based binding
HUE029357T2 (en) 2012-05-08 2017-02-28 Adaptive Biotechnologies Corp Compositions and method for measuring and calibrating amplification bias in multiplexed pcr reactions
US20150132754A1 (en) 2012-05-14 2015-05-14 Cb Biotechnologies, Inc. Method for increasing accuracy in quantitative detection of polynucleotides
SG11201407901PA (en) 2012-05-21 2015-01-29 Fluidigm Corp Single-particle analysis of particle populations
CN104508492B (en) 2012-05-25 2018-07-27 北卡罗来纳-查佩尔山大学 Microfluidic device, solid support and correlation technique for reagent
AU2013273953B2 (en) 2012-06-15 2018-12-13 The Board Of Regents Of The University Of Texas System High throughput sequencing of multiple transcripts of a single cell
EP2861761A1 (en) 2012-06-15 2015-04-22 Adaptive Biotechnologies Corporation Uniquely tagged rearranged adaptive immune receptor genes in a complex gene set
EP2861787B1 (en) 2012-06-18 2017-09-20 Nugen Technologies, Inc. Compositions and methods for negative selection of non-desired nucleic acid sequences
US20150011396A1 (en) 2012-07-09 2015-01-08 Benjamin G. Schroeder Methods for creating directional bisulfite-converted nucleic acid libraries for next generation sequencing
JP6285929B2 (en) 2012-07-17 2018-02-28 カウンシル，インコーポレーテッド System and method for detecting genetic variation
WO2014015098A1 (en) 2012-07-18 2014-01-23 Siemens Healthcare Diagnostics Inc. A method of normalizing biological samples
SG11201500313YA (en) 2012-07-24 2015-02-27 Sequenta Inc Single cell analysis using sequence tags
US10870099B2 (en) 2012-07-26 2020-12-22 Illumina, Inc. Compositions and methods for the amplification of nucleic acids
EP3578669B1 (en) 2012-08-08 2024-07-10 F. Hoffmann-La Roche AG Increasing dynamic range for identifying multiple epitopes in cells
US10273541B2 (en) 2012-08-14 2019-04-30 10X Genomics, Inc. Methods and systems for processing polynucleotides
US20150005199A1 (en) 2012-08-14 2015-01-01 10X Technologies, Inc. Compositions and methods for sample processing
US20140378349A1 (en) 2012-08-14 2014-12-25 10X Technologies, Inc. Compositions and methods for sample processing
US20150005200A1 (en) 2012-08-14 2015-01-01 10X Technologies, Inc. Compositions and methods for sample processing
US9951386B2 (en) 2014-06-26 2018-04-24 10X Genomics, Inc. Methods and systems for processing polynucleotides
US10752949B2 (en) 2012-08-14 2020-08-25 10X Genomics, Inc. Methods and systems for processing polynucleotides
US9701998B2 (en) 2012-12-14 2017-07-11 10X Genomics, Inc. Methods and systems for processing polynucleotides
US20140378345A1 (en) 2012-08-14 2014-12-25 10X Technologies, Inc. Compositions and methods for sample processing
US10584381B2 (en) 2012-08-14 2020-03-10 10X Genomics, Inc. Methods and systems for processing polynucleotides
CN113528634A (en) 2012-08-14 2021-10-22 10X基因组学有限公司 Microcapsule compositions and methods
US10221442B2 (en) 2012-08-14 2019-03-05 10X Genomics, Inc. Compositions and methods for sample processing
US20140378322A1 (en) 2012-08-14 2014-12-25 10X Technologies, Inc. Compositions and methods for sample processing
CN104884605B (en) 2012-08-24 2018-05-18 耶鲁大学 For the systems, devices and methods of high-throughput multi detection
EP3901280A1 (en) 2012-10-17 2021-10-27 Spatial Transcriptomics AB Methods and product for optimising localised or spatial detection of gene expression in a tissue sample
GB201218909D0 (en) 2012-10-22 2012-12-05 Univ Singapore Assay for the parallel detection of biological material based on PCR
CN107090491A (en) 2012-11-05 2017-08-25 鲁比康基因组学公司 Bar coding nucleic acid
EP3567116A1 (en) 2012-12-14 2019-11-13 10X Genomics, Inc. Methods and systems for processing polynucleotides
WO2014108850A2 (en) 2013-01-09 2014-07-17 Yeda Research And Development Co. Ltd. High throughput transcriptome analysis
US9683230B2 (en) 2013-01-09 2017-06-20 Illumina Cambridge Limited Sample preparation on a solid support
WO2014116729A2 (en) 2013-01-22 2014-07-31 The Board Of Trustees Of The Leland Stanford Junior University Haplotying of hla loci with ultra-deep shotgun sequencing
EP2948252A1 (en) 2013-01-24 2015-12-02 SABIC Global Technologies B.V. Microwell plate made from a polyester-polycarbonate
US10017761B2 (en) 2013-01-28 2018-07-10 Yale University Methods for preparing cDNA from low quantities of cells
EP2951557B1 (en) 2013-02-01 2022-10-05 Becton Dickinson and Company Methods and systems for assessing sample behavior in a flow cytometer
WO2014124046A1 (en) 2013-02-08 2014-08-14 Bio-Rad Laboratories, Inc. Affinity-based partition assay for detection of target molecules
KR102190198B1 (en) 2013-02-08 2020-12-14 10엑스 제노믹스, 인크. Polynucleotide barcode generation
WO2014126937A1 (en) 2013-02-14 2014-08-21 The Board Of Trustees Of The Leland Stanford Junior University Suspension arrays and multiplexed assays based thereon
US9621600B2 (en) 2013-02-26 2017-04-11 PortAura Group Method and system for providing recommendations using location information
US20140274811A1 (en) 2013-03-14 2014-09-18 Lyle J. Arnold Methods for Amplifying a Complete Genome or Transcriptome
US9328382B2 (en) 2013-03-15 2016-05-03 Complete Genomics, Inc. Multiple tagging of individual long DNA fragments
US9255265B2 (en) 2013-03-15 2016-02-09 Illumina, Inc. Methods for producing stranded cDNA libraries
WO2014145458A1 (en) 2013-03-15 2014-09-18 Arizona Board Of Regents On Behalf Of Arizona State University Nucleic acid-tagged compositions and methods for multiplexed protein-protein interaction profiling
GB2584364A (en) 2013-03-15 2020-12-02 Abvitro Llc Single cell bar-coding for antibody discovery
EP3611262B1 (en) 2013-03-15 2020-11-11 Lineage Biosciences, Inc. Methods of sequencing the immune repertoire
WO2014165762A1 (en) 2013-04-05 2014-10-09 Raindance Technologies, Inc. Rare cell analysis after negative selection
CA2909861C (en) 2013-04-25 2022-12-06 Firefly Bioworks, Inc. Multiplexed analysis of target nucleic acids
US10822605B2 (en) 2013-05-03 2020-11-03 Gensinta, Inc. Method and apparatus for producing sequence verified DNA
EP2805769A1 (en) 2013-05-24 2014-11-26 European Molecular Biology Laboratory Methods for nano-scale single cell analysis
EP3008201B1 (en) 2013-06-12 2019-08-07 The General Hospital Corporation Methods for multiplexed detection of target molecules and uses thereof
WO2014201273A1 (en) 2013-06-12 2014-12-18 The Broad Institute, Inc. High-throughput rna-seq
WO2014204939A2 (en) 2013-06-17 2014-12-24 Kim Lewis Methods for quantitative determination of protein-nucleic acid interactions in complex mixtures
EP4234716A3 (en) 2013-06-25 2023-12-06 Prognosys Biosciences, Inc. Methods for determining spatial patterns of biological targets in a sample
CA2915499A1 (en) 2013-06-27 2014-12-31 10X Genomics, Inc. Compositions and methods for sample processing
US9708657B2 (en) 2013-07-01 2017-07-18 Adaptive Biotechnologies Corp. Method for generating clonotype profiles using sequence tags
US20160153973A1 (en) 2013-07-09 2016-06-02 Lucas David Smith Device and method of rapid linker mediated label-based immunoassays
GB2525104B (en) 2013-08-28 2016-09-28 Cellular Res Inc Massively Parallel Single Cell Nucleic Acid Analysis
US10395758B2 (en) 2013-08-30 2019-08-27 10X Genomics, Inc. Sequencing methods
EP3041957A4 (en) 2013-09-04 2017-03-29 Fluidigm Corporation Proximity assays for detecting nucleic acids and proteins in a single cell
GB201317301D0 (en) 2013-09-30 2013-11-13 Linnarsson Sten Method for capturing and encoding nucleic acid from a plurality of single cells
ES2875998T3 (en) 2013-09-30 2021-11-11 Vesicode Ab Procedures for profiling molecular complexes using proximity-dependent barcodes
JP2017504307A (en) 2013-10-07 2017-02-09 セルラー リサーチ， インコーポレイテッド Method and system for digitally counting features on an array
EP3058092B1 (en) 2013-10-17 2019-05-22 Illumina, Inc. Methods and compositions for preparing nucleic acid libraries
US9709479B2 (en) 2013-10-25 2017-07-18 Massachusetts Institute Of Technology Method and apparatus for tracking cell identity
WO2015061844A1 (en) 2013-10-30 2015-05-07 The University Of Sydney Assay to stratify cancer patients
WO2015070037A2 (en) 2013-11-08 2015-05-14 Prognosys Biosciences, Inc. Polynucleotide conjugates and methods for analyte detection
SG10201807112XA (en) 2013-12-30 2018-09-27 Atreca Inc Analysis of nucleic acids associated with single cells using nucleic acid barcodes
EP3099820A4 (en) 2014-01-27 2018-01-03 The General Hospital Corporation Methods of preparing nucleic acids for sequencing
US20150218620A1 (en) 2014-02-03 2015-08-06 Integrated Dna Technologies, Inc. Methods to capture and/or remove highly abundant rnas from a heterogenous rna sample
ES2741740T3 (en) 2014-03-05 2020-02-12 Adaptive Biotechnologies Corp Methods that use synthetic molecules that contain random nucleotide segments
EP3132059B1 (en) 2014-04-17 2020-01-08 Adaptive Biotechnologies Corporation Quantification of adaptive immune cell genomes in a complex mixture of cells
US20150298091A1 (en) 2014-04-21 2015-10-22 President And Fellows Of Harvard College Systems and methods for barcoding nucleic acids
CA2947426C (en) 2014-04-29 2020-01-07 Illumina, Inc. Multiplexed single cell gene expression analysis using template switch and tagmentation
US10975371B2 (en) 2014-04-29 2021-04-13 Illumina, Inc. Nucleic acid sequence analysis from single cells
EP3901278A1 (en) 2014-05-19 2021-10-27 William Marsh Rice University Allele-specific amplification using a composition of overlapping non-allele-specific primer and allele-specific blocker oligonucleotides
EP3161157B1 (en) 2014-06-24 2024-03-27 Bio-Rad Laboratories, Inc. Digital pcr barcoding
EP3161160B1 (en) 2014-06-26 2021-10-13 10X Genomics, Inc. Methods of analyzing nucleic acids from individual cells or cell populations
WO2015200869A1 (en) 2014-06-26 2015-12-30 10X Genomics, Inc. Analysis of nucleic acid sequences
EP3626834B1 (en) 2014-07-15 2022-09-21 Qiagen Sciences, LLC Semi-random barcodes for nucleic acid analysis
WO2016040446A1 (en) 2014-09-10 2016-03-17 Good Start Genetics, Inc. Methods for selectively suppressing non-target sequences
SG11201702060VA (en) 2014-09-15 2017-04-27 Abvitro Inc High-throughput nucleotide library sequencing
US9529672B2 (en) 2014-09-25 2016-12-27 Everspin Technologies Inc. ECC word configuration for system-level ECC compatibility
IL299976B1 (en) 2014-10-17 2024-07-01 Illumina Cambridge Ltd Contiguity preserving transposition
US20180320241A1 (en) 2014-12-19 2018-11-08 Roche Sequencing Solutions, Inc. Methods for identifying multiple epitopes in selected sub-populations of cells
EP3248018B1 (en) 2015-01-22 2020-01-08 Becton, Dickinson and Company Devices and systems for molecular barcoding of nucleic acid targets in single cells
US10760120B2 (en) 2015-01-23 2020-09-01 Qiagen Sciences, Llc High multiplex PCR with molecular barcoding
EP3253479B1 (en) 2015-02-04 2022-09-21 The Regents of The University of California Sequencing of nucleic acids via barcoding in discrete entities
WO2016130578A1 (en) 2015-02-09 2016-08-18 10X Genomics, Inc. Systems and methods for determining structural variation and phasing using variant call data
EP3259371B1 (en) 2015-02-19 2020-09-02 Becton, Dickinson and Company High-throughput single-cell analysis combining proteomic and genomic information
ES2906221T3 (en) 2015-02-27 2022-04-13 Becton Dickinson Co Methods for barcoding nucleic acids for sequencing
WO2016138496A1 (en) 2015-02-27 2016-09-01 Cellular Research, Inc. Spatially addressable molecular barcoding
CN107614700A (en) 2015-03-11 2018-01-19 布罗德研究所有限公司 Genotype and phenotype coupling
US20160266094A1 (en) 2015-03-13 2016-09-15 University Of Iowa Research Foundation Cellular barcode
WO2016149418A1 (en) 2015-03-18 2016-09-22 Cellular Research, Inc. Methods and compositions for labeling targets and haplotype phasing
WO2016160965A1 (en) 2015-03-30 2016-10-06 Rubicon Genomics, Inc. Methods and compositions for repair of dna ends by multiple enzymatic activities
US11535882B2 (en) 2015-03-30 2022-12-27 Becton, Dickinson And Company Methods and compositions for combinatorial barcoding
WO2016168825A1 (en) 2015-04-17 2016-10-20 Centrillion Technology Holdings Corporation Methods for performing spatial profiling of biological molecules
WO2016172373A1 (en) 2015-04-23 2016-10-27 Cellular Research, Inc. Methods and compositions for whole transcriptome amplification
US9618871B2 (en) 2015-04-28 2017-04-11 Kyocera Document Solutions Inc. Image forming apparatus
JP2018516569A (en) 2015-05-22 2018-06-28 シグマ−アルドリッチ・カンパニー・リミテッド・ライアビリティ・カンパニーＳｉｇｍａ−Ａｌｄｒｉｃｈ Ｃｏ．， ＬＬＣ Methods and related compositions and kits for next generation genome walking
EP3303587A4 (en) 2015-05-28 2018-11-21 Kaarel Krjutskov Blocking oligonucleotides
US11124823B2 (en) 2015-06-01 2021-09-21 Becton, Dickinson And Company Methods for RNA quantification
US11248262B2 (en) 2015-08-24 2022-02-15 Qiagen Gmbh Method for generating a RNA-sequencing library
CN115928221A (en) 2015-08-28 2023-04-07 Illumina公司 Single cell nucleic acid sequence analysis
EP3347465B1 (en) 2015-09-11 2019-06-26 Cellular Research, Inc. Methods and compositions for nucleic acid library normalization
US11156611B2 (en) 2015-09-24 2021-10-26 Abvitro Llc Single cell characterization using affinity-oligonucleotide conjugates and vessel barcoded polynucleotides
US11092607B2 (en) 2015-10-28 2021-08-17 The Board Institute, Inc. Multiplex analysis of single cell constituents
CA3004310A1 (en) 2015-11-04 2017-05-11 Atreca, Inc. Combinatorial sets of nucleic acid barcodes for analysis of nucleic acids associated with single cells
US20170136458A1 (en) 2015-11-18 2017-05-18 Wafergen, Inc. Systems and methods for pooling samples from multi-well devices
US20190040381A1 (en) 2015-12-04 2019-02-07 St. Jude Children's Research Hospital, Inc. Cloning and expression system for t-cell receptors
EP3397764A4 (en) 2015-12-30 2019-05-22 Bio-Rad Laboratories, Inc. Digital protein quantification
US20170205404A1 (en) 2016-01-19 2017-07-20 General Electric Company Multifunctional beads and methods of use for capturing rare cells
WO2017139690A1 (en) 2016-02-11 2017-08-17 10X Genomics, Inc. Cell population analysis using single nucleotide polymorphisms from single cell transcriptomes
WO2017164936A1 (en) 2016-03-21 2017-09-28 The Broad Institute, Inc. Methods for determining spatial and temporal gene expression dynamics in single cells
CN109312331B (en) 2016-04-01 2022-08-02 贝勒医学院 Method for whole transcriptome amplification
EP3452614B1 (en) 2016-05-02 2023-06-28 Becton, Dickinson and Company Accurate molecular barcoding
US20190161743A1 (en) 2016-05-09 2019-05-30 President And Fellows Of Harvard College Self-Targeting Guide RNAs in CRISPR System
US10301677B2 (en) 2016-05-25 2019-05-28 Cellular Research, Inc. Normalization of nucleic acid libraries
WO2017205691A1 (en) 2016-05-26 2017-11-30 Cellular Research, Inc. Molecular label counting adjustment methods
TWI600309B (en) 2016-05-28 2017-09-21 Hon Hai Prec Ind Co Ltd Angle adjusting mechanism
US10202641B2 (en) 2016-05-31 2019-02-12 Cellular Research, Inc. Error correction in amplification of samples
US10640763B2 (en) 2016-05-31 2020-05-05 Cellular Research, Inc. Molecular indexing of internal sequences
LT3263715T (en) 2016-06-28 2020-06-25 Hifibio Method for transcriptome analysis of single cells
WO2018017949A1 (en) 2016-07-22 2018-01-25 Verily Life Sciences Llc Quantitative massively parallel proteomics
WO2018020489A1 (en) 2016-07-24 2018-02-01 Yeda Research And Development Co. Ltd. Methods and kits for analyzing dna binding moieties attached to dna
US20180037942A1 (en) 2016-08-03 2018-02-08 Cellular Research, Inc. Enzyme-independent molecular indexing
CA3033506A1 (en) 2016-08-10 2018-02-15 President And Fellows Of Harvard College Methods of de novo assembly of barcoded genomic dna fragments
CA3034924A1 (en) 2016-09-26 2018-03-29 Cellular Research, Inc. Measurement of protein expression using reagents with barcoded oligonucleotide sequences
JPWO2018061699A1 (en) 2016-09-29 2019-06-24 富士フイルム株式会社 Method for designing primers for multiplex PCR
WO2018061695A1 (en) 2016-09-29 2018-04-05 富士フイルム株式会社 Method for designing primers for multiplex pcr
KR102650753B1 (en) 2016-10-01 2024-03-26 버클리 라잇츠, 인크. DNA barcode compositions and methods for in situ identification in microfluidic devices
WO2018075693A1 (en) 2016-10-19 2018-04-26 10X Genomics, Inc. Methods and systems for barcoding nucleic acid molecules from individual cells or cell populations
CN108473975A (en) 2016-11-17 2018-08-31 领星生物科技（上海）有限公司 The system and method for detecting tumor development
EP3551768B1 (en) 2016-12-12 2024-03-06 Grail, LLC Methods for tagging and amplifying rna template molecules for preparing sequencing libraries
US10011872B1 (en) 2016-12-22 2018-07-03 10X Genomics, Inc. Methods and systems for processing polynucleotides
US20190177800A1 (en) 2017-12-08 2019-06-13 10X Genomics, Inc. Methods and compositions for labeling cells
US10550429B2 (en) 2016-12-22 2020-02-04 10X Genomics, Inc. Methods and systems for processing polynucleotides
MX2019007444A (en) 2016-12-22 2019-08-16 Guardant Health Inc Methods and systems for analyzing nucleic acid molecules.
GB201622222D0 (en) 2016-12-23 2017-02-08 Cs Genetics Ltd Reagents and methods for molecular barcoding of nucleic acids of single cells
WO2018132635A1 (en) 2017-01-12 2018-07-19 Massachusetts Institute Of Technology Methods for analyzing t cell receptors and b cell receptors
US20180208975A1 (en) 2017-01-20 2018-07-26 Merck Sharp & Dohme Corp. Assay for simultaneous genomic and proteomic analysis
CN117512066A (en) 2017-01-30 2024-02-06 10X基因组学有限公司 Method and system for droplet-based single cell bar coding
US11319583B2 (en) 2017-02-01 2022-05-03 Becton, Dickinson And Company Selective amplification using blocking oligonucleotides
FI3583214T3 (en) 2017-02-02 2023-12-19 New York Genome Center Inc Methods and compositions for identifying or quantifying targets in a biological sample
WO2018170515A1 (en) 2017-03-17 2018-09-20 The Broad Institute, Inc. Methods for identifying and modulating co-occurant cellular phenotypes
JP2020513826A (en) 2017-03-24 2020-05-21 ナショナル ユニヴァーシティー オブ シンガポール Multiplex detection method for molecules
JP7169290B2 (en) 2017-03-24 2022-11-10 ベクトン・ディキンソン・アンド・カンパニー Composite multiplets for determining multiplets
CN111051526A (en) 2017-05-23 2020-04-21 生捷科技控股公司 Method for performing analysis of spatial distribution of biomolecules
JP2020522243A (en) 2017-05-23 2020-07-30 プレジデント アンド フェローズ オブ ハーバード カレッジ Multiplexed end-tagging amplification of nucleic acids
KR102550778B1 (en) 2017-05-26 2023-07-03 에이비비트로 엘엘씨 High-throughput polynucleotide library sequencing and transcriptome analysis
US10400235B2 (en) 2017-05-26 2019-09-03 10X Genomics, Inc. Single cell analysis of transposase accessible chromatin
AU2018277019A1 (en) 2017-05-29 2019-12-19 President And Fellows Of Harvard College A method of amplifying single cell transcriptome
AU2018281745B2 (en) 2017-06-05 2022-05-19 Becton, Dickinson And Company Sample indexing for single cells
WO2019055852A1 (en) 2017-09-15 2019-03-21 Apton Biosystems, Inc. Heterogeneous single cell profiling using molecular barcoding
JP7241069B2 (en) 2017-09-25 2023-03-16 フレッド ハッチンソン キャンサー センター Highly efficient targeted in situ genome-wide profiling
ES2969957T3 (en) 2017-09-25 2024-05-23 Becton Dickinson Co Immune Receptor Barcode Bug Fixes
WO2019076768A1 (en) 2017-10-16 2019-04-25 Tervisetehnoloogiate Arenduskeskus As Method and kit for dna library preparation
WO2019084046A1 (en) 2017-10-23 2019-05-02 The Broad Institute, Inc. Single cell cellular component enrichment from barcoded sequencing libraries
WO2019099906A1 (en) 2017-11-16 2019-05-23 The Regents Of The University Of Colorado, A Body Corporate Methods to measure functional heterogeneity among single cells
EP3720605A1 (en) 2017-12-07 2020-10-14 Massachusetts Institute Of Technology Single cell analyses
WO2019113499A1 (en) 2017-12-07 2019-06-13 The Broad Institute, Inc. High-throughput methods for identifying gene interactions and networks
WO2019113506A1 (en) 2017-12-07 2019-06-13 The Broad Institute, Inc. Methods and compositions for multiplexing single cell and single nuclei sequencing
CN110462060B (en) 2017-12-08 2022-05-03 10X基因组学有限公司 Methods and compositions for labeling cells
WO2019118355A1 (en) 2017-12-12 2019-06-20 10X Genomics, Inc. Systems and methods for single cell processing
CN118547046A (en) 2017-12-22 2024-08-27 10X基因组学有限公司 Systems and methods for processing nucleic acid molecules from one or more cells
PT3735470T (en) 2018-01-05 2024-01-31 Billiontoone Inc Quality control templates for ensuring validity of sequencing-based assays
US20210213413A1 (en) 2018-02-05 2021-07-15 The Board Of Trustees Of The Leland Stanford Junior University Systems and methods for multiplexed measurements in single and ensemble cells
CN112005115A (en) 2018-02-12 2020-11-27 10X基因组学有限公司 Methods of characterizing multiple analytes from a single cell or cell population
US20210046482A1 (en) 2018-03-12 2021-02-18 Silicon Valley Scientific, Inc. Method and apparatus for processing tissue and other samples encoding cellular spatial position information
WO2019204560A1 (en) 2018-04-18 2019-10-24 The Regents Of The University Of California Method to connect chromatin accessibility and transcriptome
WO2019213237A1 (en) 2018-05-03 2019-11-07 Becton, Dickinson And Company Molecular barcoding on opposite transcript ends
US11773441B2 (en) 2018-05-03 2023-10-03 Becton, Dickinson And Company High throughput multiomics sample analysis
CN110499251A (en) 2018-05-17 2019-11-26 中国科学院大连化学物理研究所 The extracellular vesica analysis chip of the unicellular source property of high flux multiparameter and application
CN112689673A (en) 2018-07-11 2021-04-20 埃克斯基因美国公司 Transposome-enabled DNA/RNA sequencing (TED RNA-SEQ)
US20200040379A1 (en) 2018-08-03 2020-02-06 Cellular Research, Inc. Nuclei barcoding and capture in single cells
WO2020033164A1 (en) 2018-08-08 2020-02-13 Vanderbilt University Systems and methods for simultaneous detection of antigens and antigen specific antibodies
EP3837378B1 (en) 2018-08-17 2023-04-05 Becton, Dickinson and Company Aptamer barcoding
EP3844299A1 (en) 2018-08-28 2021-07-07 Becton Dickinson and Company Sample multiplexing using carbohydrate-binding and membrane-permeable reagents
WO2020072380A1 (en) 2018-10-01 2020-04-09 Cellular Research, Inc. Determining 5' transcript sequences
US11932849B2 (en) 2018-11-08 2024-03-19 Becton, Dickinson And Company Whole transcriptome analysis of single cells using random priming
CN113195717A (en) 2018-12-13 2021-07-30 贝克顿迪金森公司 Selective extension in single cell whole transcriptome analysis
EP3899030A2 (en) 2018-12-17 2021-10-27 Natera, Inc. Methods for analysis of circulating cells
ES2945227T3 (en) 2019-01-23 2023-06-29 Becton Dickinson Co Antibody Associated Oligonucleotides
JP7485681B2 (en) 2019-01-28 2024-05-16 ベクトン・ディキンソン・アンド・カンパニー Oligonucleotide-containing cellular component binding reagents and methods of use thereof
US12071617B2 (en) 2019-02-14 2024-08-27 Becton, Dickinson And Company Hybrid targeted and whole transcriptome amplification
US11965208B2 (en) 2019-04-19 2024-04-23 Becton, Dickinson And Company Methods of associating phenotypical data and single cell sequencing data
SG10201904897UA (en) 2019-05-30 2020-12-30 Nat Univ Singapore Method, structures and system for nucleic acid sequence topology assembly for multiplexed profiling of proteins.
WO2021011433A1 (en) 2019-07-12 2021-01-21 New York Genome Center, Inc Methods and compositions for scalable pooled rna screens with single cell chromatin accessibility profiling
US20210039582A1 (en) 2019-08-07 2021-02-11 ZF Passive Safety Systems US Inc. Airbag package with improved packaging
JP7522189B2 (en) 2019-11-08 2024-07-24 ベクトン・ディキンソン・アンド・カンパニー Use of Random Priming to Obtain Full-Length V(D)J Information for Immune Repertoire Sequencing
WO2021142233A1 (en) 2020-01-10 2021-07-15 10X Genomics, Inc. Methods for determining a location of a target nucleic acid in a biological sample
EP4090764A1 (en) 2020-01-13 2022-11-23 Becton, Dickinson and Company Cell capture using du-containing oligonucleotides
EP4090763A1 (en) 2020-01-13 2022-11-23 Becton Dickinson and Company Methods and compositions for quantitation of proteins and rna
EP4090762A1 (en) 2020-01-17 2022-11-23 Becton, Dickinson and Company Methods and compositions for single cell secretomics
US20210230583A1 (en) 2020-01-29 2021-07-29 Becton, Dickinson And Company Barcoded wells for spatial mapping of single cells through sequencing
WO2021155284A1 (en) 2020-01-31 2021-08-05 Becton Dickinson And Company Mesophilic dna polymerase extension blockers
CN115087746A (en) 2020-02-12 2022-09-20 贝克顿迪金森公司 Intracellular AbSeq
EP4106769A4 (en) 2020-02-17 2024-03-27 Universal Sequencing Technology Corporation Methods of barcoding nucleic acid for detection and sequencing
CN116157533A (en) 2020-02-21 2023-05-23 10X基因组学有限公司 Capturing genetic targets using hybridization methods
EP4114965A1 (en) 2020-03-05 2023-01-11 Biolegend, Inc. Methods and compositions for detecting targets
WO2021247593A1 (en) 2020-06-02 2021-12-09 Becton, Dickinson And Company Oligonucleotides and beads for 5 prime gene expression assay
US11932901B2 (en) 2020-07-13 2024-03-19 Becton, Dickinson And Company Target enrichment using nucleic acid probes for scRNAseq
EP4179110A1 (en) 2020-07-13 2023-05-17 Becton, Dickinson and Company Cdna spike-in control for single cell analysis
WO2022026909A1 (en) 2020-07-31 2022-02-03 Becton, Dickinson And Company Single cell assay for transposase-accessible chromatin
CN116529389A (en) 2020-08-20 2023-08-01 贝克顿迪金森公司 Blood cell lysing agent for separating bacteria from blood cultures
WO2022108946A1 (en) 2020-11-17 2022-05-27 Becton, Dickinson And Company Combined analysis of cell-free nucleic acids and single cells for oncology diagnostics
WO2022109339A1 (en) 2020-11-20 2022-05-27 Becton, Dickinson And Company Use of dextramer in single cell analysis
EP4247967A1 (en) 2020-11-20 2023-09-27 Becton, Dickinson and Company Profiling of highly expressed and lowly expressed proteins
US20220178909A1 (en) 2020-12-09 2022-06-09 Becton, Dickinson And Company Multiplexed single cell immunoassay
CN114015755B (en) 2020-12-31 2024-03-01 中国科学院北京基因组研究所(国家生物信息中心) Methods and kits for labeling nucleic acid molecules
EP4274907A1 (en) 2021-01-08 2023-11-15 Cellanome, Inc. Devices and methods for analyzing biological samples

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050019776A1 (en) * 2002-06-28 2005-01-27 Callow Matthew James Universal selective genome amplification and universal genotyping system
US20080299609A1 (en) * 2007-03-12 2008-12-04 Sungkyunkwan University Foundation For Corporate Collaboration Uracil-DNA glycosylase of psychrobacter sp. HJ147 and use thereof
US20110294689A1 (en) * 2010-05-27 2011-12-01 Affymetrix, Inc Multiplex Amplification Methods

Also Published As

Publication number Publication date
EP2820158B1 (en) 2018-01-10
EP3321378A1 (en) 2018-05-16
WO2013130674A1 (en) 2013-09-06
US20150133319A1 (en) 2015-05-14
GB201408829D0 (en) 2014-07-02
SG11201405274WA (en) 2014-10-30
ES2663234T3 (en) 2018-04-11
JP6375230B2 (en) 2018-08-15
AU2013226081A1 (en) 2014-09-18
CA2865575C (en) 2024-01-16
GB201319347D0 (en) 2013-12-18
JP2015511819A (en) 2015-04-23
CA2865575A1 (en) 2013-09-06
AU2013226081B2 (en) 2018-06-14
CN104364392B (en) 2018-05-25
EP2820158A1 (en) 2015-01-07
EP2820158A4 (en) 2015-08-19
US20210230582A1 (en) 2021-07-29
GB2513024B (en) 2016-08-31
GB2504240A (en) 2014-01-22
US10941396B2 (en) 2021-03-09
US11634708B2 (en) 2023-04-25
AU2018220004A1 (en) 2018-09-06
GB2504240B (en) 2015-05-27
AU2018220004B2 (en) 2020-07-02
HK1194109A1 (en) 2014-10-10
ES2904816T3 (en) 2022-04-06
EP3321378B1 (en) 2021-11-10
JP2018064585A (en) 2018-04-26
US20210238588A1 (en) 2021-08-05
CA3218231A1 (en) 2013-09-06
CN104364392A (en) 2015-02-18
JP6632596B2 (en) 2020-01-22
HK1206069A1 (en) 2015-12-31

Similar Documents

Publication Publication Date Title
GB2513024A (en) Compositions and kits for molecular counting
US11845979B2 (en) Spatial transcriptomics for antigen-receptors
US8497071B2 (en) Isolation of unknown rearranged T-cell receptors from single cells
JP5081144B2 (en) Composition for use in bacterial identification
EP3980559A1 (en) Methods for detecting immune cell dna and monitoring immune system
JP2019180415A (en) Methods of sequencing nucleic acids in mixtures and compositions related thereto
EP3714064B1 (en) Methods and kits for amplification of double stranded dna
EP3674415A1 (en) Determining antigen-specific t-cells and b-cells
US20040121313A1 (en) Methods for rapid detection and identification of bioagents in organs for transplantation
US20180282808A1 (en) Determining wt-1 specific t cells and wt-1 specific t cell receptors (tcrs)
AU2015209103B2 (en) Isothermal methods and related compositions for preparing nucleic acids
US20210024920A1 (en) Integrative DNA and RNA Library Preparations and Uses Thereof
US20220002802A1 (en) Compositions and methods for immune repertoire sequencing
CA3024355A1 (en) Methods for identification of samples
JP2023519919A (en) Assays to detect pathogens
US20220073983A1 (en) Compositions and methods for immune repertoire sequencing
US20110256541A1 (en) Compositions for use in identification of bacteria
US20230416810A1 (en) Compositions and methods for immune repertoire monitoring
CA3135619A1 (en) Methods and systems to characterize tumors and identify tumor heterogeneity
US20230340602A1 (en) Compositions and methods for immune repertoire monitoring
US20200325522A1 (en) Method and systems to characterize tumors and identify tumor heterogeneity
WO2021202861A1 (en) Method of characterisation

Legal Events

Date Code Title Description
732E Amendments to the register in respect of changes of name or changes affecting rights (sect. 32/1977)

Free format text: REGISTERED BETWEEN 20201126 AND 20201202